Sequence conversion by single strand oligonucleotide donors via non-homologous end joining in mammalian cells.

Double strand breaks (DSBs) can be repaired by homology independent nonhomologous end joining (NHEJ) pathways involving proteins such as Ku70/80, DNAPKcs, Xrcc4/Ligase 4, and the Mre11/Rad50/Nbs1 (MRN) complex. DSBs can also be repaired by homology-dependent pathways (HDR), in which the MRN and CtIP nucleases produce single strand ends that engage homologous sequences either by strand invasion or strand annealing. The entry of ends into HDR pathways underlies protocols for genomic manipulation that combine site-specific DSBs with appropriate informational donors. Most strategies utilize long duplex donors that participate by strand invasion. Work in yeast indicates that single strand oligonucleotide (SSO) donors are also active, over considerable distance, via a single strand annealing pathway. We examined the activity of SSO donors in mammalian cells at DSBs induced either by a restriction nuclease or by a targeted interstrand cross-link. SSO donors were effective immediately adjacent to the break, but activity declined sharply beyond approximately 100 nucleotides. Overexpression of the resection nuclease CtIP increased the frequency of SSO-mediated sequence modulation distal to the break site, but had no effect on the activity of an SSO donor adjacent to the break. Genetic and in vivo competition experiments showed that sequence conversion by SSOs in the immediate vicinity of the break was not by strand invasion or strand annealing pathways. Instead these donors competed for ends that would have otherwise entered NHEJ pathways.

DNA interstrand crosslink repair in mammalian cells: step by step.

Interstrand DNA crosslinks (ICLs) are formed by natural products of metabolism and by chemotherapeutic reagents. Work in E. coli identified a two cycle repair scheme involving incisions on one strand on either side of the ICL (unhooking) producing a gapped intermediate with the incised oligonucleotide attached to the intact strand. The gap is filled by recombinational repair or lesion bypass synthesis. The remaining monoadduct is then removed by nucleotide excision repair (NER). Despite considerable effort, our understanding of each step in mammalian cells is still quite limited. In part this reflects the variety of crosslinking compounds, each with distinct structural features, used by different investigators. Also, multiple repair pathways are involved, variably operative during the cell cycle. G(1) phase repair requires functions from NER, although the mechanism of recognition has not been determined. Repair can be initiated by encounters with the transcriptional apparatus, or a replication fork. In the case of the latter, the reconstruction of a replication fork, stalled or broken by collision with an ICL, adds to the complexity of the repair process. The enzymology of unhooking, the identity of the lesion bypass polymerases required to fill the first repair gap, and the functions involved in the second repair cycle are all subjects of active inquiry. Here we will review current understanding of each step in ICL repair in mammalian cells.

Targeted gene knock in and sequence modulation mediated by a psoralen-linked triplex-forming oligonucleotide.

Information from exogenous donor DNA can be introduced into the genome via homology-directed repair (HDR) pathways. These pathways are stimulated by double strand breaks and by DNA damage such as interstrand cross-links. We have employed triple helix-forming oligonucleotides linked to psoralen (pso-TFO) to introduce a DNA interstrand cross-link at a specific site in the genome of living mammalian cells. Co-introduction of duplex DNA with target region homology resulted in precise knock in of the donor at frequencies 2-3 orders of magnitude greater than with donor alone. Knock-in was eliminated in cells deficient in ERCC1-XPF, which is involved in recombinational pathways as well as cross-link repair. Separately, single strand oligonucleotide donors (SSO) were co-introduced with the pso-TFO. These were 10-fold more active than the duplex knock-in donor. SSO efficacy was further elevated in cells deficient in ERCC1-XPF, in contrast to the duplex donor. Resected single strand ends have been implicated as critical intermediates in sequence modulation by SSO, as well as duplex donor knock in. We asked whether there would be a competition between the donor species for these ends if both were present with the pso-TFO. The frequency of duplex donor knock in was unaffected by a 100-fold molar excess of the SSO. The same result was obtained when the homing endonuclease I-SceI was used to initiate HDR at the target site. We conclude that the entry of double strand breaks into distinct HDR pathways is controlled by factors other than the nucleic acid partners in those pathways.

Extensive sugar modification improves triple helix forming oligonucleotide activity in vitro but reduces activity in vivo.

We are developing triple helix forming oligonucleotides (TFOs) for gene targeting. Previously, we synthesized bioactive TFOs containing 2'-O-methylribose (2'-OMe) and 2'-O-aminoethylribose (2'-AE) residues. Active TFOs contained four contiguous 2'-AE residues and formed triplexes with high thermal stability and rapid association kinetics. In an effort to further improve bioactivity, we synthesized three series of TFOs containing the 2'-AE patch and additional ribose modifications distributed throughout the remainder of the oligonucleotide. These were either additional 2'-AE residues, the conformationally locked BNA/LNA ribose with a 2'-O,4'-C-methylene bridge, or the 2'-O,4'-C-ethylene analogue (ENA). The additionally modified TFOs formed triplexes with greater thermal stability than the reference TFO, and some had improved association kinetics. However, the most active TFOs in the biochemical and biophysical assays were the least active in the bioassay. We measured the thermal stability of triplexes formed by the TFOs in each series on duplex targets containing a change in sequence at a single position. The Tm value of the variant sequence triplexes increased as the number of all additional modifications increased. A simple explanation for the failure of the improved TFOs in the bioassay was that the increased affinity for nonspecific targets lowered the effective nuclear concentration. Enhancement of TFO bioactivity will require chemical modifications that improve interaction with the specific targets while retaining selectivity against mismatched sequences.

A human neuronal tissue culture model for Lesch-Nyhan disease.

Mutations in the gene encoding the purine salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HPRT) cause Lesch-Nyhan disease, a neurodevelopmental disorder characterized by cognitive, neurological, and behavioral abnormalities. Despite detailed knowledge of the enzyme's function, the key pathophysiological changes that accompany loss of purine recycling are unclear. To facilitate delineating the consequences of HPRT deficiency, four independent HPRT-deficient sublines of the human dopaminergic neuroblastoma, SK-N-BE(2) M17, were isolated by targeted mutagenesis with triple helix-forming oligonucleotides. As a group, these HPRT-deficient cells showed several significant abnormalities: (i) impaired purine recycling with accumulation of hypoxanthine, guanine, and xanthine, (ii) reduced guanylate energy charge and GTP:GDP ratio, but normal adenylate energy charge and no changes in any adenine nucleotide ratios, (iii) increased levels of UTP and NADP+, (iv) reduced DOPA decarboxylase, but normal monoamines, and (v) reduction in cell soma size. These cells combine the analytical power of multiple lines and a human, neuronal origin to provide an important tool to investigate the pathophysiology of HPRT deficiency.

Targeted cross-linking of the human beta-globin gene in living cells mediated by a triple helix forming oligonucleotide.

Triple helix forming oligonucleotides (TFOs) may have utility as gene targeting reagents for "in situ" gene therapy of genetic disorders. Triplex formation is challenged by negative charge repulsion between third strand and duplex phosphates, and destabilizing positive charge repulsion between adjacent protonated cytosines within pyrimidine motif third strands. Here we describe the synthesis of TFOs designed to target a site in the human beta-globin gene, which is the locus for mutations that underlie the beta-globinopathies, including sickle cell anemia. The target is an uninterrupted polypurine:polypyrimidine sequence, containing four adjacent cytosines, next to a psoralen cross-link site. Pyrimidine motif TFOs that contained four adjacent cytosines or 5-methylcytosines did not form stable triplexes at physiological pH, despite the introduction of otherwise stabilizing base and sugar analogues. We synthesized a series of pso-TFOs containing 2'-O-methyl (OMe) and 2'-O-aminoethoxy substitutions (AE), as well as 8-oxo-adenine (A8) and 2'-O-methylpseudoisocytidine (P) as neutral cytosine replacements. Thermal stability measurements indicated that TFOs with A8 did not meet criteria established in previous work. However, TFOs with P did form triplexes with appropriate T(m) and k(ON) values. A pso-TFO with AE and P residues was sufficiently active to permit the determination of targeting in living cells by direct measurement of cross-link formation at the target site. Our results validate the modification format described in our previous studies and indicate that P substitutions are an effective solution to the problem of targeting genomic sequences containing adjacent cytosines.

Triplex targeted genomic crosslinks enter separable deletion and base substitution pathways.

We have synthesized triple helix forming oligonucleotides (TFOs) that target a psoralen (pso) interstrand crosslink to a specific chromosomal site in mammalian cells. Mutagenesis of the targeted crosslinks results in base substitutions and deletions. Identification of the gene products involved in mutation formation is important for developing practical applications of pso-TFOs, and may be informative about the metabolism of other interstrand crosslinks. We have studied mutagenesis of a pso-TFO genomic crosslink in repair proficient and deficient cells. Deficiencies in non homologous end joining and mismatch repair do not influence mutation patterns. In contrast, the frequency of base substitutions is dependent on the activity of ERCC1/XPF and polymerase zeta, but independent of other nucleotide excision repair (NER) or transcription coupled repair (TCR) genes. In NER/TCR deficient cells the frequency of deletions rises, indicating that in wild-type cells NER/TCR functions divert pso-TFO crosslinks from processes that result in deletions. We conclude that targeted pso-TFO crosslinks can enter genetically distinct mutational routes that resolve to base substitutions or deletions.

The development of bioactive triple helix-forming oligonucleotides.

We are developing triple helix-forming oligonucleotides (TFOs) as gene targeting reagents in mammalian cells. We have described psoralen-conjugated TFOs containing 2'-O-methyl (2'OMe) and 2'-O-aminoethoxy (AE) ribose substitutions. TFOs with a cluster of 3-4 AE residues, with all other sugars as 2'OMe, were bioactive in a gene knockout assay in mammalian cells. In contrast, TFOs with one or two clustered, or three dispersed, AE residues were inactive. Thermal stability analysis of the triplexes indicated that there were only incremental differences between the active and inactive TFOs. However the active and inactive TFOs could be distinguished by their association kinetics. The bioactive TFOs showed markedly greater on-rates than the inactive TFOs. It appears that the on-rate is a better predictor of TFO bioactivity than thermal stability. Our data are consistent with a model in which a cluster of 3-4 AE residues stabilizes the nucleation event that precedes formation of a complete triplex. It is likely that triplexes in cells are much less stable than triplexes in vitro probably as a result of elution by chromatin-associated translocases and helicases. Consequently the biologic assay will favor TFOs that can bind and rebind genomic targets quickly.

Werner syndrome protein 1367 variants and disposition towards coronary artery disease in Caucasian patients.

The leading causes of death for individuals with Werner syndrome (WS) are myocardial infarction (MI) and stroke. The WS gene encodes a nuclear protein with both helicase and exonuclease activities. While individuals with WS have mutations that result in truncated, inactive proteins, several sequence variants have been described in apparently unaffected individuals. Some of these gene polymorphisms encode non-conservative amino acid substitutions, and it is expected that the changes would affect enzyme activity, although this has not been determined. Two research groups have studied the Cys/Arg 1367 polymorphism (located near the nuclear localization signal) in healthy and MI patients. Their results suggest that the Arg allele is protective against MI. We have characterized the Cys (C) and Arg (R) forms of the protein and find no notable difference in helicase and nuclease activities, or in nuclear/cytoplasmic distribution. The frequency of the C/R alleles in healthy individuals and subjects with coronary artery disease (CAD) drawn from the Baltimore Longitudinal Study of Aging (BLSA) was also examined. There was no indication that the R allele was protective against CAD. We conclude that the C/R polymorphism does not affect enzyme function or localization and does not influence CAD incidence in the BLSA cohort.

Importance of clustered 2'-O-(2-aminoethyl) residues for the gene targeting activity of triple helix-forming oligonucleotides.

We are developing triple helix-forming oligonucleotides (TFOs) as gene targeting reagents in living mammalian cells. We have described psoralen-linked TFOs with 2'-O-methyl and 2'-O-(2-aminoethyl) (2'-AE) substitutions that are active in a gene knockout assay in cultured cells. The assay is based on mutagenesis by psoralen, a photoactive DNA cross-linker. Previous work showed that TFOs with three or four 2'-AE residues were disproportionately more active than those with one or two substitutions. Here we demonstrate that for optimal bioactivity the 2'-AE residues must be clustered rather than dispersed. We have further characterized bioactive and inactive TFOs in an effort to identify biochemical and biophysical correlates of biological activity. While thermal stability is a standard monitor of TFO biophysical activity, we find that T(m) values do not distinguish bioactive and inactive TFOs. In contrast, measurements of TFO association rates appear to correlate well with bioactivity, in that triplex formation occurs disproportionately faster with the TFOs containing three or four 2'-AE residues. We asked if extending the incubation time prior to photoactivation would enhance the bioactivity of a TFO with a slow on rate relative to the TFO with a faster association rate. However, there was no change in bioactivity differential. These results are compatible with a model in which TFO binding in vivo is followed by relatively rapid elution by cellular functions, similar to that described for transcription factors. Under these circumstances, TFOs with faster on rates would be favored because they would be more likely to be in triplexes at the time of photoactivation.

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.

Cell cycle modulation of gene targeting by a triple helix-forming oligonucleotide.

Successful gene-targeting reagents must be functional under physiological conditions and must bind chromosomal target sequences embedded in chromatin. Triple helix-forming oligonucleotides (TFOs) recognize and bind specific sequences via the major groove of duplex DNA and may have potential for gene targeting in vivo. We have constructed chemically modified, psoralen-linked TFOs that mediate site-specific mutagenesis of a chromosomal gene in living cells. Here we show that targeting efficiency is sensitive to the biology of the cell, specifically, cell cycle status. Targeted mutagenesis was variable across the cycle with the greatest activity in S phase. This was the result of differential TFO binding as measured by cross-link formation. Targeted cross-linking was low in quiescent cells but substantially enhanced in S phase cells with adducts in approximately 20-30% of target sequences. 75-80% of adducts were repaired faithfully, whereas the remaining adducts were converted into mutations (>5% mutation frequency). Clones with mutations could be recovered by direct screening of colonies chosen at random. These results demonstrate high frequency target binding and target mutagenesis by TFOs in living cells. Successful protocols for TFO-mediated manipulation of chromosomal sequences are likely to reflect a combination of appropriate oligonucleotide chemistry and manipulation of the cell biology.

Gene targeting by triple helix-forming oligonucleotides.

Effective gene targeting reagents would have widespread utility for genomic manipulation including transgenic cell and animal construction and for gene therapy. They would also be useful in basic research as probes of chromatin structure, and as tools for studying the repair and mutagenesis of targeted DNA damage. We are developing triple helix-forming oligonucleotides (TFOs) for gene targeting in living mammalian cells. Challenges to TFO bioactivity include the impediments to the biochemistry of triplex formation presented by the physiological environment and the charge repulsion between the duplex and the third strand. In addition, there are biological constraints to target access imposed by mammalian chromatin structure. Here we describe the oligonucleotide modification format that appears to support biological activity of TFOs. In addition we show that manipulation of the cell biology, specifically the cell cycle, has a dramatic influence on TFO bioactivity.

Minimum number of 2'-O-(2-aminoethyl) residues required for gene knockout activity by triple helix forming oligonucleotides.

Triple helix forming oligonucleotides (TFOs) that bind chromosomal targets in living cells may become tools for genome manipulation, including gene knockout, conversion, or recombination. However, triplex formation by DNA third strands, particularly those in the pyrimidine motif, requires nonphysiological pH and Mg(2+) concentration, and this limits their development as gene-targeting reagents. Recent advances in oligonucleotide chemistry promise to solve these problems. For this study TFOs containing 2'-O-methoxy (2'-OMe) and 2'-O-(2-aminoethyl) (2'-AE) ribose substitutions in varying proportion have been constructed. The TFOs were linked to psoralen and designed to target and mutagenize a site in the hamster HPRT gene. T(m) analyses showed that triplexes formed by these TFOs were more stable than the underlying duplex, regardless of 2'-OMe/2'-AE ratio. However, TFOs with 2'-AE residues were more stable in physiological pH than those with only 2'-OMe sugars, as a simple function of 2'-AE content. In contrast, gene knockout assays revealed a threshold requirement--TFOs with three or four 2'-AE residues were at least 10-fold more active than the TFO with two 2'-AE residues. The HPRT knockout frequencies with the most active TFOs were 300-400-fold above the background, whereas there was no activity against the APRT gene, a monitor of nonspecific mutagenesis.

Novel mycophenolic adenine bis(phosphonate) analogues as potential differentiation agents against human leukemia.

Novel mycophenolic adenine dinucleotide (MAD) analogues have been prepared as potential inhibitors of inosine monophosphate dehydrogenase (IMPDH). MAD analogues resemble nicotinamide adenine dinucleotide binding at the cofactor binding domain of IMPDH; however, they cannot participate in hydride transfer and therefore inhibit the enzyme. The methylenebis(phosphonate) analogues C2-MAD and C4-MAD were obtained by coupling 2',3'-O-isopropylideneadenosine 5'-methylenebis(phosphonate) (22) with mycophenolic alcohols 20 and 21 in the presence of diisopropylcarbodiimide followed by deprotection. C2-MAD was also prepared by coupling of mycophenolic methylenebis(phosphonate) derivative 30 with 2',3'-O-isopropylideneadenosine. Compound 30 was conveniently synthesized by the treatment of benzyl-protected mycophenolic alcohol 27 with a commercially available methylenebis(phosphonic dichloride) under Yoshikawa's reaction conditions. C2-MAD and C4-MAD were found to inhibit the growth of K562 cells (IC(50) = 0.7 microM and IC(50) = 0.1 microM, respectively) as potently as mycophenolic acid (IC(50) = 0.3 microM). In addition, C2-MAD and C4-MAD triggered vigorous differentiation of K562 cells an order of magnitude more potently than tiazofurin, and MAD analogues were resistant to glucuronidation in vitro. These results show that C2-MAD and C4-MAD may be of therapeutic interest in the treatment of human leukemias.