Reduction of gene repair by selenomethionine with the use of single-stranded oligonucleotides

Background  

The repair of single base mutations in mammalian genes can be directed by single-stranded oligonucleotides in a process known as targeted gene repair. The mechanism of this reaction is currently being elucidated but likely involves a pairing step in which the oligonucleotide align in homologous register with its target sequence and a correction step in which the mutant base is replaced by endogenous repair pathways. This process is regulated by the activity of various factors and proteins that either elevate or depress the frequency at which gene repair takes place.     

In vivo gene repair of point and frameshift mutations directed by chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides

Synthetic oligonucleotides have been used to direct base exchange and gene repair in a variety of organisms. Among the most promising vectors is chimeric oligonucleotide (CO), a double-stranded, RNA–DNA hybrid molecule folded into a double hairpin conformation: by using the cell’s DNA repair machinery, the CO directs nucleotide exchange as episomal and chromosomal DNA. Systematic dissection of the CO revealed that the region of contiguous DNA bases was the active component in the repair process, especially when the single-stranded ends were protected against nuclease attack. Here, the utility of this vector is expanded into Saccharomyces cerevisiae. An episome containing a mutated fusion gene encoding hygromycin resistance and eGFP expression was used as the target for repair. Substitution, deletion and insertion mutations were corrected with different frequencies by the same modified single-stranded vector as judged by growth in the presence of hygromycin and eGFP expression. A substitution mutation was repaired the most efficiently followed by insertion and finally deletion mutants. A strand bias for gene repair was also observed; vectors designed to direct the repair of nucleotide on the non-transcribed (non-template) strand displayed a 5–10-fold higher level of activity. Expanding the length of the oligo-vector from 25 to 100 nucleotides increases targeting frequency up to a maximal level and then it decreases. These results, obtained in a genetically tractable organism, contribute to the elucidation of the mechanism of targeted gene repair.     

Gene targeting in mouse embryos mediated by RecA and modified single-stranded oligonucleotides

Gene targeting is a precise manipulation of endogenous gene by introduction of exogenous DNA and has contributed greatly to the elucidation of gene functions. Conventional gene targeting has been achieved through a use of embryonic stem cells. However, such procedure is often long, tedious, and expensive. This study was carried out to develop a simple procedure of gene targeting using E. coli recombinase A (RecA) and modified single-stranded oligonucleotides. The new procedure was attempted to modify X-linked hypoxanthine phosphoribosyltransferase (HPRT) gene in mouse embryos. The single-stranded oligonucleotide to target an exon 3 of HPRT was 74 bases in length including phosphorothioate linkages at each terminus to be resistant against exonucleases when introduced into zygotes. The oligonucleotide sequence was homologous to the target gene except a single nucleotide that induces a mismatch between an introduced oligonucleotide and endogenous HPRT gene. Endogenous repairing of such mismatch would give rise to the conversion of TAT to TAG stop codon thereby losing the function of the target gene. Before an introduction into zygotes, single-stranded oligonucleotides were bound to RecA to enhance the homologous recombination. The RecA–oligonucleotide complex was microinjected into the pronucleus of zygote. Individual microinjected embryos developed to the blastocyst stage were analyzed for the expected nucleotide conversion using polymerase chain reaction (PCR) and subsequent sequencing. The conversion of TAT to TAG stop codon was detected in three embryos among 48 tested blastocysts (6.25% in frequency). The result suggests that the gene targeting was feasible by relatively easier and direct method.     

Targeted gene correction of hprt mutations by 45 base single-stranded oligonucleotides

Targeted correction of a single base in a gene of an eucaryotic cell by specific oligonucleotides is a yet controversial technique. Here, we introduce the correction of point mutations in the hypoxanthine-guanine-phosphoribosyl-transferase (HPRT) gene as an additional model system to test targeted gene correction. In human, Hprt mutations cause Lesch-Nyhan syndrome. Using hamster V79 cells, we generated three cell lines with one hprt point mutation each. These cell lines were treated with specific single-stranded 45 base phosphothioate modified oligonucleotides and selected by HAT medium. The surviving clones were investigated for the correction of the respective hprt mutation. Treatment with the oligonucleotides was successful in repairing all three hprt mutations (hprt cDNA position 74, C --> T; position 151, C --> T; and position 400, G --> A). The correction efficiency was very low but reproducible. We suggest that this system allows one to investigate targeted gene correction in dependence on the target sequence and the oligonucleotides used.     

Targeted gene modification in mismatch-repair-deficient embryonic stem cells by single-stranded DNA oligonucleotides

Gene targeting through homologous recombination in murine embryonic stem (ES) cells is already strongly suppressed by DNA mismatch-repair (MMR)-dependent anti-recombination when targeting construct and target locus differ at <1% of the nucleotide positions. We demonstrate that MMR activity also raises a strong impediment to gene modification mediated by small synthetic DNA oligonucleotide sequences. In the absence of the DNA MMR gene MSH2, synthetic single-stranded deoxyribo-oligonucleotides can be used to site-specifically modify the ES cell genome. We show that PCR-based procedures can be used to identify and clone modified cells. By this method we have substituted a single codon in the retinoblastoma gene.     

Strand bias influences the mechanism of gene editing directed by single-stranded DNA oligonucleotides

Gene editing directed by modified single-stranded DNA oligonucleotides has been used to alter a single base pair in a variety of biological systems. It is likely that gene editing is facilitated by the direct incorporation of the oligonucleotides via replication and/or by direct conversion, most likely through the DNA mismatch repair pathway. The phenomenon of strand bias, however, as well as its importance to the gene editing reaction itself, has yet to be elucidated in terms of mechanism. We have taken a reductionist approach by using a genetic readout in Eschericha coli and a plasmid-based selectable system to evaluate the influence of strand bias on the mechanism of gene editing. We show that oligonucleotides (ODNs) designed to anneal to the lagging strand generate 100-fold greater 'editing' efficiency than 'those that anneal to' the leading strand. The majority of editing events (～70%) occur by the incorporation of the ODN during replication within the lagging strand. Conversely, ODNs that anneal to the leading strand generate fewer editing events although this event may follow either the incorporation or direct conversion pathway. In general, the influence of DNA replication is independent of which ODN is used suggesting that the importance of strand bias is a reflection of the underlying mechanism used to carry out gene editing.     

Specific targeted gene repair using single-stranded DNA oligonucleotides at an endogenous locus in mammalian cells uses homologous recombination

The feasibility of introducing point mutations in vivo using single-stranded DNA oligonucleotides (ssON) has been demonstrated but the efficiency and mechanism remain elusive and potential side effects have not been fully evaluated. Understanding the mechanism behind this potential therapy may help its development. Here, we demonstrate the specific repair of an endogenous non-functional hprt gene by a ssON in mammalian cells, and show that the frequency of such an event is enhanced when cells are in S-phase of the cell cycle. A potential barrier in using ssONs as gene therapy could be non-targeted mutations or gene rearrangements triggered by the ssON. Both the non-specific mutation frequencies and the frequency of gene rearrangements were largely unaffected by ssONs. Furthermore, we find that the introduction of a mutation causing the loss of a functional endogenous hprt gene by a ssON occurred at a similarly low but statistically significant frequency in wild type cells and in cells deficient in single strand break repair, nucleotide excision repair and mismatch repair. However, this mutation was not induced in XRCC3 mutant cells deficient in homologous recombination. Thus, our data suggest ssON-mediated targeted gene repair is more efficient in S-phase and involves homologous recombination.     

Targeted nucleotide repair of cyc1 mutations in Saccharomyces cerevisiae directed by modified single-stranded DNA oligonucleotides

Modified single-stranded DNA oligonucleotides have been used to direct base changes in the CYC1 gene of Saccharomyces cerevisiae. In this process, the oligonucleotide is believed to hybridize to the target site through the action of a DNA recombinase and, once bound, DNA repair enzymes act to excise the nucleotide, replace it, and revert the gene to wild-type status. Nucleotide exchange exhibits a strand bias as, in most cases, a higher level of base reversal appears in cells in which the oligonucleotide is designed to hybridize to the nontemplate strand. But, in one case, a higher level was observed when an oligonucleotide complementary to the transcribed strand was used. Mutant haploid and diploid strains are reverted to wild type at this locus with approximately the same frequency and all strains take up the oligonucleotide with approximately equal efficiency. Some repair preference for certain base mismatches was observed; for example, T/T and C/C mispairs exhibited the highest degree of reactivity. Finally, we demonstrate that proteins involved in DNA pairing can enhance the repair activity up to 22-fold, while others affect the reaction minimally. Taken together, these results confirm the importance and versatility of yeast as a model system to elucidate the factors regulating the frequency of nucleotide exchange directed by oligonucleotides.     

Strong binding of single-stranded DNA by stem-loop oligonucleotides.

We report that oligodeoxynucleotides which form stem-loop hairpin structures and which have pyrimidine-rich loops can form strong complexes with complementary single-stranded DNA sequences. Stem-loop oligonucleotides were constructed with a 25-nt T-rich loop and with variable Watson-Crick stems. The complexes of these oligomers with the sequence dA8 were studied by thermal denaturation. Evidence is presented that the complexes are one-to-one, bimolecular complexes in which the pyrimidine loop bases comprise the outer strands in a pyr.pur.pyr triplex, in effect chelating the purine strand in the center of the loop. Melting temperatures for the loop complexes are shown to be up to 29 degrees C higher than Watson-Crick duplex of the same length. It is shown that the presence of a stem increases stability of the triplex relative to an analogous oligomer without a stem. The effect of stem length on the stability of such a complex is examined. Such hairpin oligomers represent a new approach to the sequence-specific binding of single-stranded RNA and DNA. In addition, the finding raises the possibility that such a complex may exist in natural RNA folded sequences.     

Multiple roles for MSH2 in the repair of a deletion mutation directed by modified single-stranded oligonucleotides

The mechanism by which modified single-stranded oligonucleotides (MSSOs) direct base changes in genes is not completely understood, but there is evidence that DNA damage, repair and cell cycle checkpoint proteins are involved in the targeted nucleotide exchange (TNE) process. We are interested in the role of the mismatch repair protein, Msh2 in the correction of a frameshift mutation in both yeast and mammalian cells. We show that this protein exerts different and opposing influences on the TNE reaction in MSH2 deficient yeast compared to MSH2(-/-) mammalian cells and in wild-type cells that have RNAi silenced Msh2. Data from yeast show a 10-fold decrease in the targeting frequency whereas mammalian cells have an elevated correction frequency. These results show that in yeast this protein is required for efficient targeting and may play a role in mismatch recognition and repair. In mammalian cells, Msh2 plays a suppressive role in TNE reaction by either precluding the oligonucleotide annealing to the target gene or by maintenance of a cell cycle checkpoint induced by the MSSO itself. These results reveal that the mechanism of TNE between yeast and mammalian cells is not conserved, and demonstrate that the suppression of the TNE reaction can be bypassed using RNAi against MSH2 designed to knockdown its expression.     

Recovery of cell cycle delay following targeted gene repair by oligonucleotides

We have previously shown that activation of the homologous recombinational repair pathway leads to a block of cell division in corrected cells, possibly through the activity of checkpoint proteins Chk1 and Chk2. In this study, we examine the long-term impact of this stalling on the growth of cells that have enabled gene repair events. Using a mutated eGFP gene as an episomal reporter, we show that corrected (eGFP-positive) cells contain only a few active replication templates 2 weeks after electroporation, yet do not display an apoptotic or senescent phenotype. By 6 weeks after electroporation, cells resume active replication with a cell cycle profile that is comparable to that of the non-corrected (eGFP-negative) population. These results indicate that the initial stalling is transient and eGFP-positive cells eventually resume a normal phenotypic growth pattern, allowing for passaging and expansion in vitro.     

Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs

Targeted mutagenesis directed by oligonucleotides (ONs) is a promising method for manipulating the genome in higher eukaryotes. In this study, we have compared gene editing by different ONs on two new target sequences, the eBFP and the rd1 mutant photoreceptor βPDE cDNAs, which were integrated as single copy transgenes at the same genomic site in 293T cells. Interestingly, antisense ONs were superior to sense ONs for one target only, showing that target sequence can by itself impart strand-bias in gene editing. The most efficient ONs were short 25 nt ONs with flanking locked nucleic acids (LNAs), a chemistry that had only been tested for targeted nucleotide mutagenesis in yeast, and 25 nt ONs with phosphorothioate linkages. We showed that LNA-modified ONs mediate dose-dependent target modification and analyzed the importance of LNA position and content. Importantly, when using ONs with flanking LNAs, targeted gene modification was stably transmitted during cell division, which allowed reliable cloning of modified cells, a feature essential for further applications in functional genomics and gene therapy. Finally, we showed that ONs with flanking LNAs aimed at correcting the rd1 stop mutation could promote survival of photoreceptors in retinas of rd1 mutant mice, suggesting that they are also active in vivo.     

Enhanced infarct myocardium repair mediated by thermosensitive copolymer hydrogel-based stem cell transplantation

Mesenchymal stem cell (MSC) transplantation by intramyocardial injection has been proposed as a promising therapy strategy for cardiac repair after myocardium infarction. However, low retention and survival of grafted MSCs hinder its further application. In this study, copolymer with N-isopropylacrylamide/acrylic acid/2-hydroxylethyl methacrylate-poly(ɛ-caprolactone) ratio of 88:9.6:2.4 was bioconjugated with type I collagen to construct a novel injectable thermosensitive hydrogel. The injectable and biocompatible hydrogel-mediated MSC transplantation could enhance the grafted cell survival in the myocardium, which contributed to the increased neovascularization, decreased interstitial fibrosis, and ultimately improved heart function to a significantly greater degree than regular MSC transplantation. We suggest that this novel hydrogel has the potential for future stem cell transplantation.     

CD43 gene expression is mediated by a nuclear factor which binds pyrimidine-rich single-stranded DNA

CD43 is a leukocyte-specific surface molecule which plays an important role both in adhesion and signal transduction. We have identified a site spanning nucleotides +18 to +39 within the human CD43 gene promoter which in vitro is hypersensitive to cleavage by nuclease S1. Repeats of this region are sufficient to activate expression of a heterologous promoter in CD43-positive cell lines. Two nuclear factors, PyRo1 and PyRo2, interact with the hypersensitive site. PyRo1 is a single-stranded DNA-binding protein which binds the pyrimidine-rich sense strand. Mutation analysis demonstrates that the motif TCCCCT is critical for PyRo1 interaction. Replacement of this motif with the sequence CATATA abolishes PyRo1 binding and reduces expression of the CD43 promoter by 35% in Jurkat T lymphocytic cells and by 52% in the pre-erythroid/pre-megakaryocytic cell line K562. However, this same replacement failed to affect expression in U937 monocytic cells or in CEM T lymphocytic cells. PyRo1, therefore, exhibits cell-specific differences in its functional activity. Further analysis demonstrated that PyRo1 not only interacts with the CD43 gene promoter but also motifs present within the promoters of the CD11a, CD11b, CD11c and CD11d genes. These genes encode the α subunits of the β2 integrin family of leukocyte adhesion receptors. Deletion of the PyRo1 binding site within the CD11c gene reduced promoter activity in T lymphocytic cells by 47%. However, consistent with our analysis of the CD43 gene, the effect of this same deletion within U937 monocytic cells was less severe. That PyRo1 binds preferentially to single-stranded DNA and sequences within the CD43 and CD11 gene promoters suggests that expression of these genes is influenced by DNA secondary structure.     

Gene repair and mutagenesis mediated by chimeric RNA-DNA oligonucleotides: chimeraplasty for gene therapy and conversion of single nucleotide polymorphisms (SNPs)

Gene augmentation is an attractive and viable approach in treatment of inherited diseases, despite its limitations, such as the eliciting of host immune response, and the sustainability of gene expression. Therefore, alternative therapeutic approaches are being investigated, such as the use of chimeric RNA-DNA oligonucleotides (chimeraplasts), in which a mutated allele that already exists in an affected individual can be corrected. Although the only gene defects that can be corrected by chimeraplasty are point mutations, and the correction frequencies are variable, it has been observed that intracellular delivery of oligonucleotides is likely to be more efficient than that of plasmid DNA or viral vectors. Furthermore, corrected genes are expressed from their autologous promoters, thus ensuring correct spatial and temporal expression. Here we report on the recent progress made in the field of chimeraplasty, and the problems encountered.     

Enhancement and inhibition by 2'-O-hydroxyethyl residues of gene targeting mediated by triple helix forming oligonucleotides

Reagents that recognize and bind specific genomic sequences in living mammalian cells would have great potential for genetic manipulation, including gene knockout, strain construction, and gene therapy. Triple helix forming oligonucleotides (TFOs) bind specific sequences via the major groove, but pyrimidine motif TFOs are limited by their poor activity under physiological conditions. Base and sugar analogues that overcome many of these limitations have been described. In particular, 2'-O-modifications influence sugar pucker and third strand conformation, and have been important to the development of bioactive TFOs. Here we have analyzed the impact of 2'-O-hydroxyethyl (2'-HE) substitutions, in combination with other 2' modifications. We prepared modified TFOs conjugated to psoralen and measured targeting activity in a gene knockout assay in cultured hamster cells. We find that 2'-HE residues enhance the bioactivity of TFOs containing 2'-O-methyl (2'-OMe) modifications, but reduce the bioactivity of TFOs containing, in addition, 2'-O-aminoethyl (2'-AE) residues.     

Recognition of hairpin-containing single-stranded DNA by oligonucleotides containing internal acridine derivatives.

Oligodeoxynucleotides with an internal intercalating agent have been targeted to single-stranded sequences containing hairpin structures. The oligonucleotide binds to nonadjacent single-stranded sequences on both sides of the hairpin structure in such a way as to form a three-way junction. The acridine derivative is inserted at a position that allows it to interact with the three-way junction. The melting temperature (Tm) of complexes formed between the hairpin-containing target and oligonucleotides containing one internal acridine derivative was higher than that obtained with the same target and an unmodified oligonucleotide (DeltaTm = +13 degrees C). The internal acridine provided the oligonucleotide with a higher affinity than covalent attachment to the 5' end. Oligonucleotides could also be designed to recognize a hairpin-containing single-stranded nucleic acid by formation of Watson-Crick hydrogen bonds with a single-stranded part and Hoogsteen hydrogen bonds with the stem of the hairpin. An internal acridine derivative was introduced at the junction between the two domains, the double helix domain with Watson-Crick base pairs and the triple helix domain involving Hoogsteen base triplets in the major groove of the hairpin stem. Oligonucleotides with an internal acridine or an acridine at their 5' end have similar binding affinities for the stem-loop-containing target. The bis-modified oligonucleotide containing two acridines, one at the 5' end and one at an internal site, did not exhibit a higher affinity than the oligonucleotides with only one intercalating agent. The design of oligonucleotides with an internal intercalating agent might be of interest to control gene expression through recognition of secondary structures in single-stranded targets.     

Aggregation of RecA-derived peptides on single-stranded oligonucleotides triggered by schiff base-mediated crosslinking

We here show that single-stranded oligonucleotides containing 5-formyl-2'-deoxyuridine (fdU) can crosslink the peptides derived from the DNA binding site of RecA protein through a Schiff base formation. The ability of crosslinking of fdU-containing oligonucleotides was investigated using a series of peptides whose amino acid residues spanning the center of the RecA-derived peptide were sequentially replaced with lysine. Circular dichroism (CD) spectroscopy, gel mobility shift assay and sedimentation experiment demonstrated that crosslinking reaction proceeded efficiently only when the peptides bound to the oligonucleotides.