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Strategy for targeted correction of beta-globin gene IVS2–1 mutation in mammalian cells. ( A ) Schematic of the cellular assay, in which intron 2 of the beta-globin gene, mutated at position 1, interrupts the GFP sequence. In cell lines made from this construct, this mutation leads to an aberrant transcript. GFP expression in these cells requires reversion of this IVS2–1 G 3 A mutation, via PNA-stimulated gene correction, thereby restoring proper splicing. ( B ) Relative positions and sequences of the homopurine bis-PNA binding sites in intron 2 of the beta-globin gene. ( C ) Putative strand invasion complex formed by bis-PNA-35 at the beta-globin IVS2–35 site, resulting in a PNA/DNA/PNA triplex structure. ( D ) Sequences of the bis-PNAs used in the current study. The sequence of the 51-mer IVS2 donor DNA corresponds to the nontemplate strand (single base pair change underlined). J, pseudoisocytidine. ( E ) Gel shift assays of the bis-PNAs binding to their respective target sites in a plasmid construct.
Source publication
Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian cells via site-specific binding and creation of alte...
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... beta-globin gene, such that GFP expression requires proper splicing of this intronic sequence (8). A G3A mutation at position 1 of this intron (GFP/IVS2-1 G3A ) abolishes the normal donor splice site while activating a cryptic splice site, resulting in a GFP mRNA that retains an additional 47 nt of intron and yields no GFP protein expression (Fig. 1A). This GFP/IVS2-1 G3A construct was stably transfected into CHO cells to create a reporter cell line (CHO- GFP/IVS2-1 G3A ). The insertion was directed into a single, pre- defined locus as previously described (11). A similar cell line, containing the GFP gene interrupted by a wild-type beta-globin intron 2 (GFP/IVS2 wt ), was also ...
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... triplex formation can stimulate recombination by altering local DNA topology and triggering DNA repair activity (4,12). We identified five such sites within human beta-globin IVS2, ranging from IVS2-35 (and therefore, 34 base pairs from our mutation site of interest) to a sequence beginning at IVS2-830 (829 bp from the IVS2-1 mutation) (Fig. 1B). Bis-PNAs with ethylene glycol- containing linkers were designed to form PNA/DNA/PNA triplex- invasion complexes at these sites (Fig. 1C). For the bis-PNAs used in this study (Fig. 1D), cytosines in the Hoogsteen-bonding PNA strand were substituted with pseudoisocytosine to eliminate the requirement for N3 protonation of cytosine, ...
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... five such sites within human beta-globin IVS2, ranging from IVS2-35 (and therefore, 34 base pairs from our mutation site of interest) to a sequence beginning at IVS2-830 (829 bp from the IVS2-1 mutation) (Fig. 1B). Bis-PNAs with ethylene glycol- containing linkers were designed to form PNA/DNA/PNA triplex- invasion complexes at these sites (Fig. 1C). For the bis-PNAs used in this study (Fig. 1D), cytosines in the Hoogsteen-bonding PNA strand were substituted with pseudoisocytosine to eliminate the requirement for N3 protonation of cytosine, thereby enhancing complex formation at neutral pH (13). Lysines were also conjugated to the PNA molecules to increase solubility and to ...
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... IVS2, ranging from IVS2-35 (and therefore, 34 base pairs from our mutation site of interest) to a sequence beginning at IVS2-830 (829 bp from the IVS2-1 mutation) (Fig. 1B). Bis-PNAs with ethylene glycol- containing linkers were designed to form PNA/DNA/PNA triplex- invasion complexes at these sites (Fig. 1C). For the bis-PNAs used in this study (Fig. 1D), cytosines in the Hoogsteen-bonding PNA strand were substituted with pseudoisocytosine to eliminate the requirement for N3 protonation of cytosine, thereby enhancing complex formation at neutral pH (13). Lysines were also conjugated to the PNA molecules to increase solubility and to enhance strand invasion by the molecules ...
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... assess bis-PNA binding to target sites, gel shift assays were carried out. Increasing concentrations of the various bis-PNAs were incubated with plasmids containing the relevant PNA binding sites (Fig. 1E). As shown in Fig. 1E, the PNAs can strand invade into and bind to their target sites in the plasmid substrate in vitro at concentrations as low as 200 nM. The multiple bands seen in the gel shift assay result from structural or stoichiometric isomers formed by bis-PNAs binding to duplex DNA (15). These isomers are formed at high PNA ...
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... assess bis-PNA binding to target sites, gel shift assays were carried out. Increasing concentrations of the various bis-PNAs were incubated with plasmids containing the relevant PNA binding sites (Fig. 1E). As shown in Fig. 1E, the PNAs can strand invade into and bind to their target sites in the plasmid substrate in vitro at concentrations as low as 200 nM. The multiple bands seen in the gel shift assay result from structural or stoichiometric isomers formed by bis-PNAs binding to duplex DNA (15). These isomers are formed at high PNA concentrations in ...
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... second intron and thereby introduce a thalassemia-like muta- tion (sequence change shown in Fig. 4A). We found that treatment of K562 or beta-YAC BMCs with this HBB donor DNA alone does lead to low-level genomic modification that is specifically detected by an allele-specific forward primer [ Fig. 4B and see supporting information (SI) Fig. S1 for validation of assay specificity]. As controls, mock transfected cells show no PCR amplification using mutant-specific forward primers, indicating a lack of gene modifi- cation in these cells, whereas the use of wild-type-specific primers yields amplification products from all treated cell populations as expected, because only a ...
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... products using the primers specific for the introduced mutation (Fig. 4B). The specificity of the bis-PNA effect was demonstrated by the use of a control bis-PNA that does not bind the beta-globin gene; when transfected with HBB donor DNA, this control bis-PNA fails to stimulate gene modifi- cation beyond that mediated by donor DNA alone (Fig. S1C). We chose bis-PNA-194 for these studies because it induced the highest relative frequencies of gene correction in our CHO reporter assay. The K562 cells were transfected during S phase and treated with chloroquine posttransfection to enhance oligonucleotide uptake and delivery, whereas the beta-YAC BMCs were transfected as an ...
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... The sequences of the bis-PNAs used in this study are shown in Fig. 1D. Bis-PNAs with 8-amino-2,6-dioxaoctanoic acid linkers were either syn- The modification is a 6 nt substitution (underlined) that introduces a splicing mutation in the beta-globin gene. The HBB donor DNA sequence corresponds to the template strand of the target site. (B) K562 cells synchronized in S phase, or beta-YAC BMCs, were ...
Citations
... Later, it was shown that the triple helix formed upon the binding of PNA to DNA stimulates recombination in the cell (co-transfection of TFOs and short, single-stranded DNA donor molecules), [140]. Chin and his co-authors proposed the use of TFO PNAs to induce a frameshift mutation in the same β-globin gene (responsible for β-talassemia) to restore proper splicing of the gene [141]. They improved this approach further in 2012 to correct the hemoglobin gene mutations in hematopoietic cell precursors reaching the probability of correction to 1.63% with PNA and donor DNA versus 0.29% spontaneous correction with donor DNA alone [142]. ...
Originally discovered by Nielsen in 1991, peptide nucleic acids and other artificial genetic polymers have gained a lot of interest from the scientific community. Due to their unique biophysical features these artificial hybrid polymers are now being employed in various areas of theranostics (therapy and diagnostics). The current review provides an overview of their structure, principles of rational design, and biophysical features as well as highlights the areas of their successful implementation in biology and biomedicine. Finally, the review discusses the areas of improvement that would allow their use as a new class of therapeutics in the future.
... 317 The gPNA binds at a mixed-sequence site on the gene via the DNA duplex invasion, allowing the gene edit to be performed without the need of the triplex-forming sequence typically required for the aegPNAbased gene-editing. 318 Although the efficiency still requires improvement to compete with the well-developed CRISPR-Cas9 and related technologies, 319 such PNA-based gene editing appears to offer promising potential for therapeutics and beyond. However, despite the seemingly solid evidence of gene-editing by gPNA including in vivo 320 and in utero 321 studies in mouse models, a question has been raised whether the observed effects are real or just artifacts resulting from the possible aggregation of the (miniPEG) PNA onto the donor DNA. ...
Peptide nucleic acid or PNA is a synthetic DNA mimic that contains a sequence of nucleobases attached to a peptide-like backbone derived from N-2-aminoethylglycine. The semi-rigid PNA backbone acts as a scaffold that arranges the nucleobases in a proper orientation and spacing so that they can pair with their complementary bases on another DNA, RNA, or even PNA strand perfectly well through the standard Watson-Crick base-pairing. The electrostatically neutral backbone of PNA contributes to its many unique properties that make PNA an outstanding member of the xeno-nucleic acid family. Not only PNA can recognize its complementary nucleic acid strand with high affinity, but it does so with excellent specificity that surpasses the specificity of natural nucleic acids and their analogs. Nevertheless, there is still room for further improvements of the original PNA in terms of stability and specificity of base-pairing, direction of binding, and selectivity for different types of nucleic acids, among others. This review focuses on attempts towards the rational design of new generation PNAs with superior performance by introducing conformational constraints such as a ring or a chiral substituent in the PNA backbone. A large collection of conformationally rigid PNAs developed during the past three decades are analyzed and compared in terms of molecular design and properties in relation to structural data if available. Applications of selected modified PNA in various areas such as targeting of structured nucleic acid targets, supramolecular scaffold, biosensing and bioimaging, and gene regulation will be highlighted to demonstrate how the conformation constraint can improve the performance of the PNA. Challenges and future of the research in the area of constrained PNA will also be discussed.
... Different chemically modified ONs, such as locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and 2 -O-Methyl RNA are being used to modify aberrant splicing patterns. SSOs have been successfully applied to modulate splicing patterns in the context of various splicing disorders, such as for spinal muscular atrophy and Duchenne muscular dystrophy [16][17][18]. These findings emphasize the therapeutic potential of SSOs in human diseases; however, further development is necessary to enhance efficiency and systemic delivery of SSOs. ...
Splice-switching therapy with splice-switching oligonucleotides (SSOs) has recently proven to be a clinically applicable strategy for the treatment of several mis-splice disorders. Despite this, wider application of SSOs is severely limited by the inherently poor bioavailability of SSO-based therapeutic compounds. Cell-penetrating peptides (CPPs) are a class of drug delivery systems (DDSs) that have recently gained considerable attention for improving the uptake of various oligonucleotide (ON)-based compounds, including SSOs. One strategy that has been successfully applied to develop effective CPP vectors is the introduction of various lipid modifications into the peptide. Here, we repurpose hydrocarbon-modified amino acids used in peptide stapling for the orthogonal introduction of hydrophobic modifications into the CPP structure during peptide synthesis. Our data show that α,α-disubstituted alkenyl-alanines can be successfully utilized to introduce hydrophobic modifications into CPPs to improve their ability to formulate SSOs into nanoparticles (NPs), and to mediate high delivery efficacy and tolerability both in vitro and in vivo. Conclusively, our results offer a new flexible approach for the sequence-specific introduction of hydrophobicity into the structure of CPPs and for improving their delivery properties.
... Most notably, significant progress in gene editing of hematopoietic stem cells has been reported by Glazer's team in collaboration with other groups [297]. Glazer and co-workers have been studying triplex-forming bis-PNAs as gene mutagenesis and editing tools for more than two decades [285,310,311]. ...
Peptide nucleic acid (PNA) is arguably one of the most successful DNA mimics, despite a most dramatic departure from the native structure of DNA. The present review summarizes 30 years of research on PNA's chemistry, optimization of structure and function, applications as probes and diagnostics, and attempts to develop new PNA therapeutics. The discussion starts with a brief review of PNA's binding modes and structural features, followed by the most impactful chemical modifications, PNA enabled assays and diagnostics, and discussion of the current state of development of PNA therapeutics. While many modifications have improved on PNA's binding affinity and specificity, solubility and other biophysical properties, the original PNA is still most frequently used in diagnostic and other in vitro applications. Development of therapeutics and other in vivo applications of PNA has notably lagged behind and is still limited by insufficient bioavailability and difficulties with tissue specific delivery. Relatively high doses are required to overcome poor cellular uptake and endosomal entrapment, which increases the risk of toxicity. These limitations remain unsolved problems waiting for innovative chemistry and biology to unlock the full potential of PNA in biomedical applications.
... (5) Consequently, a few places have used this approach as a definitive treatment, in spite of the fact that the graftversus-host disease have a potential longterm complication of allogeneic hematopoietic stem cells (HSCs) transplantation. (6) Moreover, accessibility of allogeneic bone marrow is restricted by finding an identical human leucocyte antigen (HLA)matched bone marrow donor. Likewise, patients with severe β-thalassemia may benefit from new genetic and cellular approches. ...
... (5) Consequently, a few places have used this approach as a definitive treatment, in spite of the fact that the graftversus-host disease have a potential longterm complication of allogeneic hematopoietic stem cells (HSCs) transplantation. (6) Moreover, accessibility of allogeneic bone marrow is restricted by finding an identical human leucocyte antigen (HLA)matched bone marrow donor. Likewise, patients with severe β-thalassemia may benefit from new genetic and cellular approches. ...
Recently, gene therapy clinical preliminaries have been effectively applied to hemoglobinopathies, for example, sickle cell disease (SCD) and β-thalassemia. Among the extraordinary disclosures that prompted the structure of genetic ways to deal with fix these disorders is the revelation of the β-globin locus control region and a few related transcription factors, which determine hemoglobin
exchanging just as significant level, erythroidspecific expression of genes at the ß-globin locus. Additionally, expanding proof shows that lentiviral vectors are proficient tools to embed large DNA components into nondividing hematopoietic stem cells, indicating consoling safe coordination profiles. On the other hand, genome altering could reestablish expression of fetal hemoglobin or target explicit mutations to restore expression of the wild-type β-globin
gene. The latest clinical preliminaries for βthalassemia and SCD are demonstrating promising results: patients had the option to stop transfusions or had diminished transfusion necessities. Be that as it may, toxic myeloablation and the significant expense of current ex vivo hematopoietic stem cell gene therapy stages speak to a barrier to a far reaching use of these methodologies. In this review, we sum up these gene therapy procedures and progressing clinical preliminaries. At last, we talk about potential systems to improve results, lessen myeloablative regimens and future difficulties to decrease the cost of gene therapy platform.
... mini-PEG chains on their backbone have been reported to effectively promote gene-editing processes without sequence restrictions [140]. Correction of the IVS2-1 (G→A) mutation in the human β-blobin gene-a genome mutation causing β-thalassemia-was originally obtained in vitro by co-transfecting a donor-DNA and some bisPNAs with a nucleofection/electroporation system [141]. Subsequently, the correction of the same genomic site was performed by delivering the best bisPNA/donor-DNA couple into CD34+ human progenitor cells with PLGA nanoparticles [142]. ...
... Further studies demonstrated the feasibility of this approach for correcting: (a) the CCR5-Δ32 mutation, involved in HIV-resistance [133,146] in different in vitro and in vivo Correction of the IVS2-1 (G→A) mutation in the human β-blobin gene-a genome mutation causing β-thalassemia-was originally obtained in vitro by co-transfecting a donor-DNA and some bisPNAs with a nucleofection/electroporation system [141]. Subsequently, the correction of the same genomic site was performed by delivering the best bisPNA/donor-DNA couple into CD34+ human progenitor cells with PLGA nanoparticles [142]. ...
... Subsequently, the correction of the same genomic site was performed by delivering the best bisPNA/donor-DNA couple into CD34+ human progenitor cells with PLGA nanoparticles [142]. Further investigations also indicated that the PLGA-based delivery system was superior to the previously reported nucleofection strategy [141], reporting a 63-fold increase of the gene-correction rate for the former internalization method. Moreover, the retention of the genome modification was reported in both erythroid-and neutrophil-differentiated cells after 30 days of culture of NPs-treated progenitors. ...
The number of applications of peptide nucleic acids (PNAs)—oligonucleotide analogs with a polyamide backbone—is continuously increasing in both in vitro and cellular systems and, parallel to this, delivery systems able to bring PNAs to their targets have been developed. This review is intended to give to the readers an overview on the available carriers for these oligonucleotide mimics, with a particular emphasis on newly developed multi-component- and multifunctional vehicles which boosted PNA research in recent years. The following approaches will be discussed: (a) conjugation with carrier molecules and peptides; (b) liposome formulations; (c) polymer nanoparticles; (d) inorganic porous nanoparticles; (e) carbon based nanocarriers; and (f) self-assembled and supramolecular systems. New therapeutic strategies enabled by the combination of PNA and proper delivery systems are discussed.
... Accordingly, the incubation of both DG44 and DG44-p11Mut cell lines with 5 µg/mL aphidicolin or 2 mM hydroxyurea for 3 h before incubation with the repair-PPRHs increased the repair frequency by 2-fold (Solé et al., 2014). This is in keeping with other studies showing increased gene correction frequencies when incubating repair oligonucleotides after treatment with hydroxyurea or aphidicolin (Parekh-Olmedo et al., 2003;Ferrara et al., 2004;Wu et al., 2005;Chin et al., 2008;Engstrom and Kmiec, 2008). ...
Monogenic disorders are often the result of single point mutations in specific genes, leading to the production of non-functional proteins. Different blood disorders such as ß-thalassemia, sickle cell disease, hereditary spherocytosis, Fanconi anemia, and Hemophilia A and B are usually caused by point mutations. Gene editing tools including TALENs, ZFNs, or CRISPR/Cas platforms have been developed to correct mutations responsible for different diseases. However, alternative molecular tools such as triplex-forming oligonucleotides and their derivatives (e.g., peptide nucleic acids), not relying on nuclease activity, have also demonstrated their ability to correct mutations in the DNA. Here, we review the Repair-PolyPurine Reverse Hoogsteen hairpins (PPRHs) technology, which can represent an alternative gene editing tool within this field. Repair-PPRHs are non-modified single-stranded DNA molecules formed by two polypurine mirror repeat sequences linked by a five-thymidine bridge, followed by an extended sequence at one end of the molecule which is homologous to the DNA sequence to be repaired but containing the corrected nucleotide. The two polypurine arms of the PPRH are bound by intramolecular reverse-Hoogsteen bonds between the purines, thus forming a hairpin structure. This hairpin core binds to polypyrimidine tracts located relatively near the target mutation in the dsDNA in a sequence-specific manner by Watson-Crick bonds, thus producing a triplex structure which stimulates recombination. This technology has been successfully employed to repair a collection of mutants of the dhfr and aprt genes within their endogenous loci in mammalian cells and could be suitable for the correction of mutations responsible for blood disorders.
... As we know, PNA can not only form duplex structures with complementary oligonucleotides, but can also form highly stable PNA 2 -DNA triplex by two homopyrimidine PNA strands and a homopurine DNA strand, in which one PNA strand forms Waston-Crick base pairs with the DNA strand and the other one forms Hoogsteen base pairs ( Figure 1C). [41][42][43] Besides, PNAs can bind DNA by strand invasion into the duplex at homopurine/ homopyrimidine stretches to form a so-called "D-loop". [44] It is also reported that a symmetrical cyanine dye containing benzothiazole groups 3,3'-diethylthiadicarbocyanine (DiSC 2 (5) in Figure 1B) can bind with high affinity to a variety of PNAcontaining hybrids, not only PNA/DNA and PNA/PNA duplexes, but also a bisPNA/DNA triplex consisting of seven base triplets. ...
Peptide nucleic acids (PNAs), the synthetic DNA mimics that can bind to oligonucleotides to form duplexes, triplexes, and quadruplexes, could be advantageous as probes for nucleic acid sequences owing to their unique physicochemical and biochemical properties. We have found that a homopurine PNA strand could bind to two homopyrimidine DNA strands to form a PNA‐DNA2 triplex. Moreover, the cyanine dye DiSC2(5) could bind with high affinity to this triplex and cause a noticeable color change. On the basis of this phenomenon, we have designed a label‐free colorimetric sensing platform for miRNAs from cancer cells by using a PNA‐DNA2 triple‐helix molecular switch (THMS) and DiSC2(5). This sensing platform can detect miRNA‐21 specifically with a detection limit of 0.18 nM, which is comparable to that of the THMS‐mediated fluorescence sensing platform. Moreover, this colorimetric platform does not involve any chemical modification or enzymatic signal amplification, which boosts its applicability and availability at the point of care in resource‐limited settings. The universality of this approach can be simply achieved by altering the sequences of the probe DNA for specific targets.
... The repair-associated DNA syntheses, in turn, lead to DNA modification within [2,3] or proximal [3] to the PNA binding site. Further, we have demonstrated that this exogenously induced but endogenously controlled DNA metabolism can result in gene disruption [2][3][4][5] due to stochastic repair events, or precise gene modification [1,[6][7][8][9][10][11][12][13][14][15][16][17] when templated by a donor DNA oligomer introduced with the PNA (Figure 1). The precision of this latter application has been leveraged by our group to correct pathologic mutations in otherwise disease-related genes [6,7,9,[15][16][17], and introduce non-natural but benign (and in some contexts, beneficial) genomic modifications [8,10,12] in normal genetic backgrounds ( Figure 1). ...
... Further, we have demonstrated that this exogenously induced but endogenously controlled DNA metabolism can result in gene disruption [2][3][4][5] due to stochastic repair events, or precise gene modification [1,[6][7][8][9][10][11][12][13][14][15][16][17] when templated by a donor DNA oligomer introduced with the PNA (Figure 1). The precision of this latter application has been leveraged by our group to correct pathologic mutations in otherwise disease-related genes [6,7,9,[15][16][17], and introduce non-natural but benign (and in some contexts, beneficial) genomic modifications [8,10,12] in normal genetic backgrounds ( Figure 1). ...
... The same bisPNA targeting strategy has been used to stimulate recombination reactions in the β-globin gene (HBB) [6,9], in which pathologic mutations underlie the primary pathophysiology of β-thalassemia and sickle cell disease. Several bisPNA oligomers, directed to different purine stretches in intron 2 (IVS2) of HBB, were shown to be useful for stimulating recombination between the gene and a donor DNA designed to correct a thalassemia associated mutation at position 1 (hence IVS2-1) of the target intron [6]. ...
Many important biological applications of peptide nucleic acids (PNAs) target nucleic acid binding in eukaryotic cells, which requires PNA translocation across at least one membrane barrier. The delivery challenge is further exacerbated for applications in whole organisms, where clearance mechanisms rapidly deplete and/or deactivate exogenous agents. We have demonstrated that nanoparticles (NPs) composed of biodegradable polymers can encapsulate and release PNAs (alone or with co-reagents) in amounts sufficient to mediate desired effects in vitro and in vivo without deleterious reactions in the recipient cell or organism. For example, poly(lactic-co-glycolic acid) (PLGA) NPs can encapsulate and deliver PNAs and accompanying reagents to mediate gene editing outcomes in cells and animals, or PNAs alone to target oncogenic drivers in cells and correct cancer phenotypes in animal models. In this chapter, we provide a primer on PNA-induced gene editing and microRNA targeting—the two PNA-based biotechnological applications where NPs have enhanced and/or enabled in vivo demonstrations—as well as an introduction to the PLGA material and detailed protocols for formulation and robust characterization of PNA/DNA-laden PLGA NPs.
