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Potentiating homologous recombination using triplex-forming peptide nucleic acids (PNAs) can be used to mediate targeted sequence editing by donor DNAs and thereby induce functional gene expression to supplant non-functional counterparts. Mutations that disrupt the normal function of the β-globin subunit cause hemoglobinopathies such as sickle cell disease and β-thalassemias. However, expression of the functional γ-globin subunit in adults, a benign condition called hereditary persistence of fetal hemoglobin (HPFH), can ameliorate the severity of these disorders, but this expression is normally silenced. Here, we harness triplex-forming PNA-induced donor DNA recombination to create HPFH mutations that increase the expression of γ-globin in adult mammalian cells, including β-yeast artificial chromosome (YAC) bone marrow and hematopoietic progenitor cells (HPCs). Transfection of human cells led to site-specific modification frequencies of 1.63% using triplex-forming PNA γ-194-3K in conjunction with donor DNAs, compared with 0.29% using donor DNAs alone. We also concurrently modified the γ-globin promoter to insert both HPFH-associated point mutations and a hypoxia-responsive element (HRE), conferring increased expression that was also regulated by oxygen tension. This work demonstrates application of oligonucleotide-based gene therapy to induce a quiescent gene promoter in mammalian cells and regulate its expression via an introduced HRE transcription factor binding site for potential therapeutic purposes.

Triplex-forming peptide nucleic acids (PNAs) are powerful gene therapy agents that can enhance recombination of short donor DNAs with genomic DNA, leading to targeted and specific correction of disease-causing genetic mutations. Therapeutic use of PNAs is severely limited, however, by challenges in intracellular delivery, particularly in clinically relevant targets such as hematopoietic stem and progenitor cells. Here, we demonstrate efficient and nontoxic PNA-mediated recombination in human CD34(+) cells using poly(lactic-co-glycolic acid) (PLGA) nanoparticles for intracellular oligonucleotide delivery. Treatment of progenitor cells with nanoparticles loaded with PNAs and DNAs targeting the β-globin locus led to levels of site-specific modification in the range of 0.5-1% in a single treatment, without detectable loss in cell viability, resulting in a 60-fold increase in modified and viable cells as compared to nucleofection. As well, the differentiation capacity of the progenitor cells treated with nanoparticles did not change relative to untreated progenitor cells, indicating that nanoparticles are safe and minimally disruptive delivery vectors for PNAs and DNAs to mediate gene modification in human primary cells. This is the first demonstration of the use of biodegradable nanoparticles to deliver genome-editing agents to human primary cells, and provides a strong rationale for systemic delivery of complex nucleic acid mixtures designed for gene correction.

Gene targeting via homologous recombination offers a potential strategy for therapeutic correction of mutations in disease-related human genes. However, there is a need to improve the efficiency of site-specific recombination by transfected donor DNAs. Oligonucleotide-mediated triple helix formation has been shown to constitute a DNA lesion sufficient to provoke DNA repair and thereby stimulate recombination. However, the ability of triplex-forming oligonucleotides (TFOs) to induce recombination between a target locus and a donor DNA has so far been demonstrated only with multicopy episomal targets in mammalian cells. Using cell lines containing the firefly luciferase reporter gene, we have now established the ability of TFOs to induce gene correction by exogenous donor DNAs at a single-copy chromosomal locus. We find that cotransfection of TFOs and short, single-stranded DNA donor molecules into mammalian cells yields gene correction in a dose-dependent manner at frequencies up to 0.1%, which is five- to ninefold above background. We demonstrate both oligonucleotide-specific and target site-specific effects. We also find that recombination can be induced by both parallel and antiparallel triple helix formation. These results provide further support for the development of TFOs as reagents to stimulate site-specific correction of defective human genes.

Top of pageAbstract Many of the mutations that lead to the blood disease |[beta]|-thalassemia are the result of single base mutations that result in a decrease or lack of |[beta]|-globin protein. The reversion of these mutations back to the wild-type sequence in genomic DNA would result in permanent restoration of globin protein expression. One mutation, G>A change which occurs at the first nucleotide of intron 2 in the |[beta]|-globin gene (designated IVS2-1 G>A), destroys the 5' donor splice site at the end of exon 2 and abolishes correct splicing of pre-mRNA. Because triplex formation via peptide nucleic acids (PNAs) has been shown to provoke repair and recombination upon binding to duplex DNA, it is believed that PNA binding can be utilized to stimulate gene conversion events. Several potential PNA target sites have been identified near the IVS2-1 G>A mutation including a polypurine run beginning 24 nt downstream from IVS2-1, a binding site which we have designated IVS2-24. Using a reporter system consisting of GFP interrupted by |[beta]|-globin IVS2, we have demonstrated restoration of reporter gene expression when cell lines containing the inactivating mutation were treated with PNAs and short donor molecules comprised of the wild-type sequence. Reporter gene expression has been confirmed by both FACS and fluorescence microscopy and was observed over a 6 week time period after a single treatment. This demonstrates a potentially powerful tool in the gene-targeting of diseases caused by single base mutations.

Top of pageAbstract Triplex forming oligonucleotides (TFOs) can induce site-specific damage, repair and recombination in episomal and chromosomal targets. However, several limitations preclude their effective use for gene targeting at the present time. For example, purine-rich TFOs have reduced binding in physiologic levels of monovalent cations, especially K+, apparently due to self-association at the guanine N7. We have substituted a 30-mer purine-rich TFO with the base analog 7-deaza-8-aza-guanine (PPG), in which the N7 and C8 positions of the guanine ring are exchanged. We show here that the PPG- substituted TFOs have increased binding affinity to a polypurine target site in vitro, even in the presence of physiologic [K+]. As well, the PPG-substituted TFOs are more efficient at forming covalent crosslinks at a chromosomally integrated target site. Notably, TFOs containing PPG substitutions at every third guanine induced almost a four-fold increase in mutation frequency in a chromosomal reporter gene in mammalian cells, almost twice the rate as a TFO containing natural guanine. In addition, we are interested in using modified TFOs to target disease-related genes in mammalian cells; preliminary results indicate that PPG-substituted TFOs can target an endogenous intronic sequence of the beta-globin locus to promote gene correction. PPG modifications, therefore, can augment triplex specificity and stability, resulting in enhanced gene targeting and correction.

Triplex-forming oligonucleotides (TFOs) bind DNA in a sequence-specific manner at polypurine/polypyrimidine sites and mediate targeted genome modification. Triplexes are formed by either pyrimidine TFOs, which bind parallel to the purine strand of the duplex (pyrimidine, parallel motif), or purine TFOs, which bind in an anti-parallel orientation (purine, anti-parallel motif). Initial studies demonstrated the ability of TFOs in both motifs to deliver mutagenic agents in a DNA site-specific manner. It was found that purine motif TFOs can induce gene modification in chromosomal DNA containing multiple copies of the mutated gene via the effect of the triple helix itself. Gene modification with TFOs includes induced recombination between a DNA target and a donor DNA molecule, a process that allows a TFO to exert an effect at a distance from the third-strand binding site. In this work, we report the ability of a N3->P5 phosphoramidate-modified pyrimidine motif TFO to induce repair and recombination in the absence of any DNA-reactive conjugate. We establish the ability of this modified TFO when co-transfected with short, single-stranded donor DNAs to induce gene correction at a single-copy chromosomal locus in mammalian cells at frequencies up to 0.1%. We demonstrate sequence, target site, and dose-dependent effects of the TFO. Additionally, we show that the induction of recombination is independent of the binding motif and the orientation of the TFO (parallel versus anti-parallel), but is dependent on the donor fragment being homologous to the transcribed strand of the duplex. This work expands the number of targets available for triplex-mediated gene targeting and shows effective targeting at a chromosomal locus.

Peptide nucleic acids (PNAs) are DNA binding molecules, which offer the potential to selectively modulate gene expression. These DNA analogues in which the purine and pyrimidine bases are attached to a polyamine backbone, maintain a spacing similar to DNA, but result in an achiral, neutrally charged molecule. PNAs can bind to DNA via Watson-Crick complementarity, with binding affinities significantly higher than those of the corresponding DNA oligomers. However, the biological activity of these potential antigene compounds has been limited by inefficient cellular uptake. Currently, the techniques used to introduce these molecules into cells, microinjection, co-mixture with cationic lipids, and electroporation, are limited by their cytotoxic effects. Here, we investigate the effect of cell-penetrating peptides on the biological activity of PNAs as measured in an assay for gene-targeted mutagenesis. Using the transport peptide derived from the third helix of the homeodomain of antennapedia (Antp), we tested PNA-peptide conjugates compared to unmodified PNAs. Confocal microscopy studies indicated increased cellular uptake of PNA when linked to Antp, consistent with the gene targeting data. Furthermore, i.p. injection of labeled PNA-Antp conjugate indicate in vivo nuclear uptake in mouse tissue. These results suggest that peptide conjugation may enhance intranuclear delivery of reagents designed to bind to chromosomal DNA, thus improving antigene potential.

Triple helix-forming oligonucleotides (TFOs) can bind to polypurine/polypyrimidine regions in DNA in a sequence-specific manner. The resulting triplexes have been shown to constitute a DNA structure that can provoke binding by the damage recognition factors, XPA and RPA, and to stimulate DNA repair via the nucleotide excision repair (NER) pathway. Based on these observations, experiments were undertaken to determine the extent to which triplex structures can stimulate recombination events within genomic DNA in a site-directed manner. Studies in which TFOs were targeted to episomal SV40 genomes in COS cells suggested that intermolecular triplexes could stimulate recombination between tandemly repeated reporter gene sequences in a pathway dependent on XPA and other NER factors. Experiments in mouse Ltk-cells containing a dual TK gene substrate demonstrated that intrachromosomal gene conversion could be provoked by high affinity triplex formation, at frequencies as high as 1-2%, if the TFOs were delivered into the cells by microinjection. Recent work in both COS cells using an episomal target and in human cell free extracts using plasmid substrates has further demonstrated that triplex formation can promote intermolecular recombination. TFOs were shown to induce recombination between a target gene and short DNA fragment, in cases where the fragment is either covalently linked to or is completely separate from the TFO. In the extracts, immunodepletion and complementation with purified proteins demonstrated a requirement for XPA and for the human recombinase, HsRad51, in the reaction. In recent work, the ability of TFOs to stimulate recombination and thereby mediate gene repair in a dose-dependent manner at a chromosomal locus in mammalian cells has been established. Overall, this work raises the possibility that DNA binding ligands such as TFOs and related molecules such as a peptide nucleic acids (PNAs) may be useful in strategies designed to mediate targeted genome modification and genetic therapy by stimulating recombination in site-specific manner.