Phosphoribosyltransferase Gene Research Articles

Cloning a second form of adenine phosphoribosyl transferase gene ( TaAPT2) from wheat and analysis of its association with thermo-sensitive genic male sterility (TGMS)

Adenine phosphoribosyl transferase (APRT) is the key enzyme that converts adenine to adenosine monophosphate (AMP) in the purine salvage pathway. A novel form of APRT gene from wheat ( Triticum aestivum L.) was cloned through a combination of bioinformatic, RT-PCR and rapid amplification of cDNA ends (RACE) strategies. The total length of the cDNA sequence is 1097 nucleotides, in which an open reading frame (ORF) was found that encodes 221 amino acid residues. Alignment of the deduced amino acid sequence and that of the other plant APRT genes indicates that the novel cDNA in full-length is the form 2 of wheat APRT gene, which was named as TaAPT2. Twelve Single Nucleotide Polymorphism (SNP) sites were identified in the TaAPT2 gene from BNY-S, one wheat temperature-sensitive genic male sterile (TGMS) mutant line derived from the wild type of BNY-F, among which a single base substitution in the purine-binding domain resulted in the Asn to Thr transition. The protein secondary structure prediction revealed that this SNP mutation subsequently resulted in many changes in the quantity and the positions of the helix, extended strand and the loop, which perhaps affect the combining efficiency of the APRT. Northern analysis indicated that the abundance of TaAPT2 gene transcript in the young spikelets reduced obviously in BNY-S under low temperature stress (lower than 10 °C) at the early stage of pollen fertility alternation, however, the transcript of TaAPT2 gene in BNY-F was not affected, indicating that wheat TaAPT2 gene may be related to the fertility alternation and abortion of pollen development.

A comparison of somatic mutational spectra in healthy study populations from Russia, Sweden and USA

A comparison of mutation spectra at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene of peripheral blood T-lymphocytes may provide an insight into the aetiology of somatic mutation contributing to carcinogenesis and other diseases. To increase the knowledge of mutation spectra in healthy people, we have analysed HPRT mutant T-cells of 50 healthy Russians originally recruited as controls in a study involving Chernobyl clean-up workers [I.M. Jones, H.Galick, P.Kato et al. (2002) Radiat. Res., 158, 424-442]. Reverse transcriptase-polymerase chain reactions and DNA sequencing identified 161 independent mutations among 176 thioguanine-resistant mutants. Forty mutations affected splicing mechanisms and 27 deletions or insertions of 1-60 nt were identified. Ninety-four single base substitutions were identified, including 62 different mutations at 55 different nucleotide positions, of which 19 had not been reported previously in human T-cells. Comparison of this base substitution spectrum with mutation spectra in a USA [K.J.Burkhart-Schultz, C.L. Thompson and I.M. Jones (1996) Carcinogenesis, 17, 1871-1883] and two Swedish populations [A.Podlutsky, A.-M.Osterholm, S.-M.Hou, A. Hofmaier and B. Lambert (1998) Carcinogenesis, 19, 557-566; A.Podlutsky, S.M.Hou, F.Nyberg, G. Pershagen and B. Lambert (1999) Mutat. Res., 431, 325-39] revealed similarity in the type, frequency and distribution of mutations in the four spectra, consistent with aetiologies inherent in human metabolism. There were 15-19 identical mutations in the three pairwise comparisons of Russian with USA and Swedish spectra. Intriguingly, there were 21 mutations unique to the Russian spectrum, and comparison by the Monte Carlo method of W.T. Adams and T.R. Skopek [(1987) J. Mol. Biol., 194, 391-396] indicated that the Russian spectrum was different from both Swedish spectra (P = 0.007, 0.002), but not different from the USA spectrum (P = 0.07) when Bonferroni correction for multiple comparisons was made (P < 0.008 required for significance). Age and smoking did not account for these differences. Other factors causing mutational differences need to be explored.

Identification of Two Novel Mutations in Adenine Phosphoribosyltransferase Gene in Patients with 2,8‐Dihydroxyadenine Urolithiasis

AbstractFor Abstract see ChemInform Abstract in Full Text.

2,8-dihydroxyadeninuria: Are there no cases in scandinavia?

Homozygosity or mixed heterozygosity for mutations in the adenine phosphoribosyltransferase gene cause enzyme deficiency directing adenine through an alternative metabolic pathway. This results in the production of 2,8-dihydroxyadenine, which is actively secreted into the urine. 2,8-dihydroxyadenine is insoluble at physiological urinary pH but as marked supersaturation is possible the manifestations differ: there may be minimal consequences, there may be infiltration of the tubulointerstitial tissue with acute or chronic damage or there may be stone formation in the urinary tract. Effective treatment can be offered and therefore the prognosis depends upon the renal function at diagnosis. Treatment consists of adequate fluid intake, a low-purine diet and administration of allopurinol. Urinary 2,8-dihydroxyadenine crystals are easily recognized under a microscope. The diagnosis of 2,8-dihydroxyadeninuria can be confirmed by estimation of adenine phosphoribosyltransferase activity in erythrocyte lysates. More than 300 cases of 2,8-dihydroxyadeninuria have been diagnosed worldwide, most of them in Japan, France and Iceland. One case has been reported in Finland but there have been no reports from the Scandinavian peninsula or from Denmark. The relevant mutations may be very rare in these countries but underdiagnosis is also possible.

Bystander effects in UV-induced genomic instability: Antioxidants inhibit delayed mutagenesis induced by ultraviolet A and B radiation

BackgroundGenomic instability is characteristic of many types of human cancer. Recently, we reported that ultraviolet radiation induced elevated mutation rates and chromosomal instability for many cell generations after ultraviolet irradiation. The increased mutation rates of unstable cells may allow them to accumulate aberrations that subsequently lead to cancer. Ultraviolet A radiation, which primarily acts by oxidative stress, and ultraviolet B radiation, which initially acts by absorption in DNA and direct damage to DNA, both produced genomically unstable cell clones. In this study, we have determined the effect of antioxidants on induction of delayed mutations by ultraviolet radiation. Delayed mutations are indicative of genomic instability.MethodsDelayed mutations in the hypoxanthine phosphoribosyl transferase (hprt) gene were detected by incubating the cells in medium selectively killing hprt mutants for 8 days after irradiation, followed by a 5 day period in normal medium before determining mutation frequencies.ResultsThe UVB-induced delayed hprt mutations were strongly inhibited by the antioxidants catalase, reduced glutathione and superoxide dismutase, while only reduced glutathione had a significant effect on UVA-induced delayed mutations. Treatment with antioxidants had only minor effects on early mutation frequenies, except that reduced glutathione decreased the UVB-induced early mutation frequency by 24 %. Incubation with reduced glutathione was shown to significantly increase the intracellular amount of reduced glutathione.ConclusionThe strong effects of these antioxidants indicate that genomic instability, which is induced by the fundamentally different ultraviolet A and ultraviolet B radiation, is mediated by reactive oxygen species, including hydrogen peroxide and downstream products. However, cells take up neither catalase nor SOD, while incubation with glutathione resulted in increased intracellular levels of glutathione. Previously, we have shown that ultraviolet induced delayed mutations may be induced via a bystander effect and that this effect is 5-fold higher for UVB radiation than for UVA radiation. Therefore, we propose that the antioxidants inhibit an ultraviolet radiation-induced bystander effect and that the effect is transmitted via the medium and via an internal transfer between cells, like gap junctional intercellular communication, for UVB radiation and only by the latter mechanism for UVA radiation.

Multiplex Polymerase Chain Reaction Analysis of UV‐A– and UV‐B–induced Delayed and Early Mutations in V79 Chinese Hamster Cells¶

ABSTRACTWe previously reported that approximately 10% of V79 Chinese hamster fibroblast populations clonally derived from single cells immediately after irradiation with either ultraviolet B (UV‐B, 290–320 nm, mainly 311 nm) or ultraviolet A (UV‐A, 320–400 nm, mainly 350–390 nm) radiation exhibit genomic instability. The instability is revealed by relatively high mutation frequencies in the hypoxanthine phosphoribosyl transferase (hprt) gene up to 23 cell generations after irradiation. These delayed mutant clones exhibited higher levels of oxidative stress than normal cells. Therefore, persistently increased oxidative stress has been proposed as a mechanism for UV‐induced genomic instability. This study investigates whether this mechanism is reflected in the deletion spectrum of delayed mutant clones. Eighty‐eight percent of the delayed mutant clones derived from UV‐A–irradiated populations were found to have total deletion of the hprt gene. Correspondingly, 81% of UV‐A–induced early mutations (i.e. detected shortly after irradiation) also had total deletions. Among delayed UV‐B–induced mutant clones, 23% had total deletions and 8% had deletion of one exon, whereas all early UV‐B events were either point mutations or small deletions or insertions. In conclusion, the multiplex polymerase chain reaction deletion screen showed that there were explicit differences in the occurrence of large gene alterations between early and delayed mutations induced by UV‐B radiation. For UV‐A radiation the deletion spectra were similar for delayed and early mutations. UV‐A radiation is, in contrast to UV‐B radiation, only weakly absorbed by DNA and probably induces mutation almost solely via production of reactive oxygen species. Therefore, the present results support the hypothesis that persistent increase in oxidative stress is involved in the mechanism of UV‐induced genomic instability.

Multiplex Polymerase Chain Reaction Analysis of UV-A– and UV-B–induced Delayed and Early Mutations in V79 Chinese Hamster Cells¶

We previously reported that approximately 10% of V79 Chinese hamster fibroblast populations clonally derived from single cells immediately after irradiation with either ultraviolet B (UV-B, 290-320 nm, mainly 311 nm) or ultraviolet A (UV-A, 320-400 nm, mainly 350-390 nm) radiation exhibit genomic instability. The instability is revealed by relatively high mutation frequencies in the hypoxanthine phosphoribosyl transferase (hprt) gene up to 23 cell generations after irradiation. These delayed mutant clones exhibited higher levels of oxidative stress than normal cells. Therefore, persistently increased oxidative stress has been proposed as a mechanism for UV-induced genomic instability. This study investigates whether this mechanism is reflected in the deletion spectrum of delayed mutant clones. Eighty-eight percent of the delayed mutant clones derived from UV-A-irradiated populations were found to have total deletion of the hprt gene. Correspondingly, 81% of UV-A-induced early mutations (i.e. detected shortly after irradiation) also had total deletions. Among delayed UV-B-induced mutant clones, 23% had total deletions and 8% had deletion of one exon, whereas all early UV-B events were either point mutations or small deletions or insertions. In conclusion, the multiplex polymerase chain reaction deletion screen showed that there were explicit differences in the occurrence of large gene alterations between early and delayed mutations induced by UV-B radiation. For UV-A radiation the deletion spectra were similar for delayed and early mutations. UV-A radiation is, in contrast to UV-B radiation, only weakly absorbed by DNA and probably induces mutation almost solely via production of reactive oxygen species. Therefore, the present results support the hypothesis that persistent increase in oxidative stress is involved in the mechanism of UV-induced genomic instability.

Somatic mutant frequency at theHPRT locus in children associated with a pediatric cancer cluster linked to exposure to two superfund sites

The somatic mutant frequency (Mf) of the hypoxanthine phosphoribosyl transferase (HPRT) gene has been widely used as a biomarker for the genotoxic effects of exposure but few studies have found an association with environmental exposures. We measured background Mfs in 49 current and former residents of Dover Township, New Jersey, who were exposed during childhood to industrially contaminated drinking water. The exposed subjects were the siblings of children who developed cancer after residing in Dover Township, where the incidence of childhood cancer has been elevated since 1979. Mfs from this exposed group were compared to Mfs in 43 age-matched, presumably unexposed residents of neighboring communities with no known water contamination and no increased cancer incidence. Statistical comparisons were based on the natural logarithm of Mf (lnMF). The mean Mf for the exposed group did not differ significantly from the unexposed group (3.90 x 10(-6) vs. 5.06 x 10(-6); P = 0.135), but unselected cloning efficiencies were higher in the exposed group (0.55 vs. 0.45; P = 0.005). After adjustment for cloning efficiency, lnMf values were very similar in both groups and age-related increases were comparable to those previously observed in healthy children. The results suggest that HPRT Mf may not be a sensitive biomarker for the genotoxic effects of environmental exposures in children, particularly when substantial time has elapsed since exposure.

331 MODIFIED SINGLE-STRANDED OLIGONUCLEOTIDE-RECOMBINASE COMPLEX MEDIATES GENE TARGETING IN MOUSE EMBRYOS

Gene targeting is an in situ manipulation of an endogenous gene in a precise manner by the introduction of exogenous DNA. The process of gene targeting involves a homologous recombination reaction between the targeted genomic sequence and an exogenous targeting vector. In elucidating the function of many genes, gene targeting has become the most important method of choice. Conventional gene targeting has been achieved through the use of embryonic stem cells. However, such a procedure is often long, tedious, and expensive and has been limited in the mouse only due to a lack of usable embryonic stem cells in other species. This study was carried out to develop a much simplified procedure of gene targeting using E. coli recombinase recA and modified single-stranded oligonucleotides. The new procedure was attempted to modify X-linked hypoxanthine phosphoribosyltransferase (HPRT) gene. The single-stranded oligonucleotide to target exon 3 of HPRT was 74 bases in length and included three phosphorothioate linkages at each terminus (also known as S-oligo) so as to be resistant against exonucleases when introduced into zygotes. The oligonucleotide sequence was homologous to the target gene except for a single nucleotide that induces a mismatch between the introduced oligonucleotide and endogenous HPRT gene. Although the exact mechanism is yet unknown, endogenous repairing of such a mismatch would give rise to the conversion of TAT to TAG stop codon, thereby losing the function of the target gene. Prior to an introduction into zygotes, modified single-stranded oligonucleotides were preincubated with recA recombinase to enhance the homologous recombination. The recA-oligonucleotide complex was microinjected into the pronuclei of zygotes. Individual microinjected embryos that developed to the blastocyst stage were analyzed for the expected nucleotide conversion using PCR and subsequent sequencing. The conversion of TAT to TAG stop codon was confirmed in two embryos among forty tested blastocysts, so that the frequency of gene targeting was approximately 5%. The result suggests that the gene targeting was feasible by this relatively easier direct method. Subsequent transfer of gene-targeted embryos to recipients to obtain transgenic mice missing the function of HPRT gene is underway. Further technical refinement and enhancement of homologous recombination frequency will be required for the practical use of this new approach for gene targeting in mice.

Lesch-Nyhan症候群患者に新しく同定された転座変異の解析

Lesch-Nyhan症候群患者に新しく同定された転座変異の解析

Identification of Two Novel Mutations in Adenine Phosphoribosyltransferase Gene in Patients with 2,8‐Dihydroxyadenine Urolithiasis

Five mutations in the adenine phosphoribosyltransferase (APRT) gene have been described in Japanese patients with APRT deficiency. We investigated the APRT gene from three patients with APRT deficiency and two novel mutations, G133D and V84M, were determined.

A survey of splice variants of the human hypoxanthine phosphoribosyl transferase and DNA polymerase beta genes: products of alternative or aberrant splicing?

Errors during the pre-mRNA splicing of metazoan genes can degrade the transmission of genetic information, and have been associated with a variety of human diseases. In order to characterize the mutagenic and pathogenic potential of mis-splicing, we have surveyed and quantified the aberrant splice variants in the human hypoxanthine phosphoribosyl transferase (HPRT) and DNA polymerase beta (POLB) in the presence and the absence of the Nonsense Mediated Decay (NMD) pathway, which removes transcripts with premature termination codons. POLB exhibits a high frequency of splice variants (40-60%), whereas the frequency of HPRT splice variants is considerably lower (approximately 1%). Treatment of cells with emetine to inactivate NMD alters both the spectrum and frequency of splice variants of POLB and HPRT. It is not certain at this point, whether POLB and HPRT splice variants are the result of regulated alternative splicing processes or the result of aberrant splicing, but it appears likely that at least some of the variants are the result of splicing errors. Several mechanisms that may contribute to aberrant splicing are discussed.

Immune responses to recombinants of the South African vaccine strain of lumpy skin disease virus generated by using thymidine kinase gene insertion

The South African vaccine strain of lumpy skin disease virus (type SA-Neethling) is currently being developed as a vector for recombinant vaccines of economically important livestock diseases throughout Africa. In this study, the feasibility of using the viral thymidine kinase gene as the site of insertion was investigated and recombinant viruses were evaluated in animal trials. Two separate recombinants were generated and selected for homogeneity expressing either the structural glycoprotein gene of bovine ephemeral fever virus (BEFV) or the two structural glycoprotein genes of Rift Valley fever virus (RVFV). Both recombinants incorporate the enhanced green fluorescent protein (EGFP) as a visual marker and the Escherichia coli guanine phosphoribosyl transferase (gpt) gene for dominant positive selection. The LSDV-RVFV recombinant construct (rLSDV-RVFV) protected mice against virulent RVFV challenge. In a small-scale BEFV-challenge cattle trial the rLSDV-BEFV construct failed to fully protect the cattle against virulent challenge, although both a humoral and cellular BEFV-specific immune response was elicited.

Mutagenicity of the mycotoxin patulin in cultured Chinese hamster V79 cells, and its modulation by intracellular glutathione

Because the ability of the mycotoxin patulin (PAT) to cause gene mutations in mammalian cells is still ambiguous, we have studied the mutagenicity of PAT at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene locus in cultured Chinese hamster V79 cells with normal, depleted, and elevated glutathione (GSH) levels. PAT was more toxic to GSH-depleted cells than to normal cells and caused an increase of the intracellular GSH level in normal and GSH-depleted cells. It also caused synchronization of the cell cycle due to a temporary accumulation of cells in the G2/M phase; this G2/M arrest was more persistent in GSH-depleted than in normal cells. PAT gave rise to a clear and concentration-dependent induction of HPRT mutations at non-cytotoxic concentrations in V79 cells with normal GSH level; the lowest PAT concentration causing a significant number of mutant cells was 0.3 micromolar, and the mutagenic potency of PAT equaled that of the established mutagen 4-nitroquinoline-N-oxide. The mutagenicity of PAT was again more pronounced, by a factor of about three, in GSH-depleted V79 cells. Elevated GSH levels abolished all observed effects of PAT. These data support the notion that PAT is a mutagenic mycotoxin, in particular in cells with low GSH concentration. The ability of PAT to cause gene mutations in mammalian cells might have a bearing on its carcinogenicity.

Genotoxicity of environmental tobacco smoke: a review

Environmental tobacco smoke (ETS), or second-hand smoke, is a widespread contaminant of indoor air in environments where smoking is not prohibited. It is a significant source of exposure to a large number of substances known to be hazardous to human health. Numerous expert panels have concluded that there is sufficient evidence to classify involuntary smoking (or passive smoking) as carcinogenic to humans. According to the recent evaluation by the International Agency for Research on Cancer, involuntary smoking causes lung cancer in never-smokers with an excess risk in the order of 20% for women and 30% for men. The present paper reviews studies on genotoxicity and related endpoints carried out on ETS since the mid-1980s. The evidence from in vitro studies demonstrates induction of DNA strand breaks, formation of DNA adducts, mutagenicity in bacterial assays and cytogenetic effects. In vivo experiments in rodents have shown that exposure to tobacco smoke, whole-body exposure to mainstream smoke (MS), sidestream smoke (SS), or their mixture, causes DNA single strand breaks, aromatic adducts and oxidative damage to DNA, chromosome aberrations and micronuclei. Genotoxicity of transplacental exposure to ETS has also been reported. Review of human biomarker studies conducted among non-smokers with involuntary exposure to tobacco smoke indicates presence of DNA adducts, urinary metabolites of carcinogens, urinary mutagenicity, SCEs and hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene mutations (in newborns exposed through involuntary smoking of the mother). Studies on human lung cancer from smokers and never-smokers involuntarily exposed to tobacco smoke suggest occurrence of similar kinds of genetic alterations in both groups. In conclusion, these overwhelming data are compatible with the current knowledge on the mechanisms of carcinogenesis of tobacco-related cancers, occurring not only in smokers but with a high biological plausibility also in involuntary smokers.

Mutagenicity evaluation of Schistosoma spp. extracts by the umu-test and V79/HGPRT gene mutation assay

Schistosomiasis has been suspected of being a risk factor for various types of cancers for sometime, e.g., bladder cancer, colorectal cancer and hepatic cancer. Among them, the etiological relationship between urinary schistosomiasis and bladder cancer is now widely accepted. However, mechanisms of the carcinogenesis are still unclear. Here, we tested the mutagenicity of the parasite extracts by the umu-test and hypoxanthine guanine phosphoribosyltransferase (HGPRT) gene mutation assay, which both overcome disadvantages of the Ames plate assay. Adult worm extracts and egg extracts of Schistosoma haematobium and Schistosoma mansoni were tested. Under our experimental conditions, neither worm nor egg extracts were shown to have any mutagenicity in both tests even in the presence of S9 mix. Our results suggest that there is very little possibility of immediate gene mutation due to the parasite-derived substances in schistosomiasis-related carcinogenesis.

Studies on the HPRT mutant frequency in T lymphocytes from healthy Indian male population as a function of age and smoking

Mutant frequency at the hypoxanthine-guanine phosphoribosyltransferase ( HPRT) gene in the peripheral blood lymphocytes obtained from 44 healthy individuals (23 non-smokers and 21 smokers) of an Indian male population was studied using T-lymphocyte cloning assay. It was found that ln MF increased with age at a rate of 2.5% per year ( P < 0.001). Blood samples from smokers showed a significant ( P < 0.037) increase in HPRT mutant frequency (MF) (10.43 ± 4.74 × 10 −6) as compared to that obtained from non-smokers (7.69 ± 3.69 × 10 −6). This study also showed a significant ( P < 0.027) inverse correlation between ln MF and non-selected cloning efficiency (CE). However, with respect to age no variation was observed in cloning efficiency. The results obtained in this study showed a good comparison with those reported in different populations of the world.

Frequency of Tk and Hprt lymphocyte mutants and bone marrow micronuclei in mice treated neonatally with zidovudine and didanosine.

The nucleoside analog zidovudine (3'-azido-3'-deoxythymidine, AZT), by itself or in combination with other anti- retroviral drugs, is used perinatally to prevent mother to child transmission of human immunodeficiency virus type 1. AZT is mutagenic in vitro and mutagenic and carcinogenic when administered to neonatal mice. A previous study indicated that the anti-retroviral agent didanosine (2',3'-dideoxyinosine, ddI) potentiated the mutagenicity of AZT in the thymidine kinase (TK) gene of cultured human TK6 lymphoblastoid cells. We have evaluated whether or not ddI affects the in vivo genotoxicity of AZT by breeding C57Bl/6N/Tk+/- female mice with C3H/HeNMTV male mice and treating the offspring daily on postnatal days 1-8 with 200 mg/kg ddI alone or in combination with 200 mg/kg AZT. One day after the last dose, bone marrow polychromatic erythrocytes (PCEs) were obtained to assess the induction of micronuclei; 3 weeks following treatment, the induction of mutants was determined in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) and Tk genes of splenic T lymphocytes from B6C3F1/Tk+/- mice. The mixture of AZT and ddI, but not ddI alone, caused a significant increase in micronucleated PCEs. When assessed 3 weeks after dosing, ddI did not induce mutations in the Hprt or Tk genes. The mixture of AZT and ddI also did not induce mutations in the Hprt gene, but did induce a significant increase in Tk mutants, similar to that observed previously with AZT alone. The induction of mutations in the Tk gene by the mixture of AZT and ddI was associated with loss of the wild-type Tk+ allele. These data indicate that, under the conditions of this experiment, ddI is not mutagenic in neonatal B6C3F1/Tk+/- mice and that it does not potentiate the mutagenicity of AZT.

Complementation of replication origin function in mouse embryonic stem cells by human DNA sequences

A functional origin of replication was mapped to the transcriptional promoter and exon 1 of the hypoxanthine–guanine phosphoribosyltransferase (HPRT) gene in the mouse and human genomes. This origin was lost in mouse embryonic stem (ES) cells with a spontaneous deletion (∼36 kb) at the 5′ end of the HPRT locus. Restoration of HPRT activity by homologous recombination with human/mouse chimeric sequences reconstituted replication origin activity in two independent ES cell lines. Quantitative PCR analyses of abundance of genetic markers in size-fractionated nascent DNA indicated that initiation of DNA replication coincided with the site of insertion in the mouse genome of the 335 bp of human DNA containing the HPRT exon 1 and a truncated promoter. The genetic information contained in the human sequence and surrounding mouse DNA was analyzed for cis-acting elements that might contribute to selection and functional activation of a mammalian origin of DNA replication.

Zinc finger nuclease-boosted gene targeting and synergistic transient regenerative gene therapy for long-term stem cell gene therapy

Stem cells have both self-renewing and homing/differentiative capabilities and thereby provide for life-long cell replacement in tissues and organs. They are therefore the ideal targets for most gene therapy protocols, i.e. both for long-term and transient gene expression protocols where they are either the long term carriers of the therapeutic gene (inherited/degenerative disease) or the mobilized/recruted targets of a transient regenerative process such as the formation of new blood vessels. Long-term gene therapy is thus amenable to synergistic combinations where ex vivo/in vivo genetic engineering of stem cells can be associated with in vivo transient topical expression of minigenes encoding various factors such as homing, regenerative and differentiative ones. Such a strategy is still hampered by many hurdles of which random integration of therapeutic DNA is a major safety concern. Emerging technologies are however aimed at efficient site-specific integration of therapeutic transgenes and at endogenous gene repair/modification. Promising site-specific integration vectors are at the pre-clinical stage and rely on an adeno-associated virus (AAV) rep platform or on phage phiC31 integrase. However, unlike these approaches, gene targeting is driven by homologous recombination and has thus target flexibility. It mediates DNA exchanges between chromosomal DNA and transfecting/transducing DNA, thereby providing the means to modify at will the sequence of target chromosomal DNA. Gene targeting stands thus as the ultimate process both for gene repair/alteration and targeted (i.e. site-specific) transgene integration. Such a process is however highly inefficient unless target chromosomal DNA is struck by a double-strand break (DSB). In addition, it is overwhelmed by random integration. In order to increase gene targeting frequency and eliminate random integration, we devised an approach that relies on the transfer into target cells of premade presynaptic filaments, i.e. the very stochiometric complexes between recombinase protein and single-stranded DNA (ssDNA) that mediate the key reaction from homologous recombination (homologous DNA pairing-strand exchange with double-stranded [ds] DNA). Upon publication of the enzymatic properties of recombinase RAD51, we invented chimeric presynaptic filaments with a dsDNA core, and shifted from gene conversion to 'true' gene targeting. Chimeric zinc finger nucleases are now available that create sequence-specific DSBs in target chromosomal DNA and stimulate gene targeting as expected. Such designed nucleases open exciting potentialities for standard gene targeting (non-viral) but also for emerging AAV gene targeting that has been shown to be boosted by DSB too. In these approaches, gene targeting frequency is raised to the random-integration level, i. e. ~1% of transfected/transduced cells. Our current approach is thus discussed in terms of synergistic combinations in which random-integration is blocked by the use of premade presynaptic complexes while homologous recombination is promoted both by premade presynaptic complexes and by sequence-specific DSB of target chromosomal DNA. Long-term gene therapy is thus amenable to sophisticated protocols in which stem-cell gene-targeting is combined with transient regenerative gene therapy, and might therefore apply to the treatment of the neurologic symptoms of the Lesch–Nyhan disease in which the target is our paragon model, the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene.