The cDNA encoding PH-20 hyaluronidase from human sperm has been mutated at five positions by in vitro mutagenesis. We have changed three acidic amino acids and two arginine residues that are conserved in the sequence of mammalian PH-20 polypeptides as well as in the hyaluronidases from bee and hornet venom. Of the former, the mutants [Gln113]PH-20 and [Gln249]PH-20 had no detectable enzymatic activity; the mutant [Asn111]PH-20 had about 3% activity. The mutant [Thr252]PH-20 was also inactive, while [Gly176]PH-20 had only about 1% activity. This indicates that the PH-20 hyaluronidases, like numerous enzymes that hydrolyze glycosidic bonds, have acidic amino acids in their active site. Moreover, for the binding of the substrate, the polyanion hyaluronan, arginine residues appear to be essential.
In this protocol, two oligonucleotides are used to prime DNA synthesis by a high-fidelity polymerase on a denatured plasmid template. The two oligonucleotides both contain the desired mutation and have the same starting and ending positions on opposite strands of the plasmid DNA. The entire lengths of both strands of the plasmid DNA are amplified in a linear fashion during several rounds of thermal cycling, generating a mutated plasmid containing staggered nicks on opposite strands. Because of the amount of template DNA used in the amplification reaction, the background of transformed colonies containing wild-type plasmid DNA can be quite high unless steps are taken to enrich for mutant molecules. In this protocol, the products of the linear amplification reaction are treated with the restriction enzyme DpnI, which specifically cleaves fully methylated GMe6ATC sequences. DpnI will therefore digest the bacterially generated DNA used as template for amplification, but it will not digest DNA synthesized during the course of the reaction in vitro. DpnI-resistant molecules, which are rich in the desired mutants, are recovered by transforming E. coli cells to antibiotic resistance. Because the method works well with virtually any plasmid of moderate size (
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The ruling[1] confirms that in-vitro mutagenesis falls under the exemption of Annex IB of the GMO Directive. With this, plant varieties resulting from in vitro mutagenesis are exempted from the obligations of Directive 2001/18.
The ECJ highlights that the exemption under Annex IB must be read in conjunction with Recital 17 which elaborates on the history of safe use of a method in several applications. Taking this into account the ECJ reasonably considers not only the mutagenesis method but also the resulting genetic modification to be decisive elements for an exemption from the GMO Directive.
This is a much more science-based approach compared to the pure process-based judgement from 2018. The Court states that those mutagenesis methods can be exempted that lead to genetic modifications of an organism which do not differ by their nature or by the rate at which they occur, from those obtained by a technique of mutagenesis which has conventionally been used in several applications and has a long safety record.
Epidemiological studies in the United States [10, 11] and Japan [12] show an association between the incidence of disease (lung cancer and respiratory disease) and long-term exposure to air pollution including SPM with a diameter below 2.5 m (PM2.5). Recent investigations conclude that outdoor air pollution is classified as IARC Group 1 [13]. However, the cancer risk of air pollutant mixtures that contain mutagenic by-products of combustion has only been evaluated on the basis of limited epidemiological data. To improve the health risk assessment of whole air pollutants, it is necessary to conduct experimental animal studies to quantitatively evaluate how the exposure to a mixture of air pollutants induces DNA damage, such as DNA adducts, that can lead to in vivo mutagenesis and potentially carcinogenesis; furthermore, it is important to know whether such air pollutants could induce mutations in germline cells.
The presence of mutagens in ambient air, especially in SPM, has been surveyed in various countries by using in vitro bioassay systems, such as the Ames test [14, 15]. Studies in Japan have shown that mutagens are ubiquitously present in air-borne particles collected in large cities [16]. Matsumoto et al. [17] reported that the contents of the PM2.5 fraction collected at an intersection with heavy traffic in Tokyo exerted higher mutagenicity than did larger air-borne particles from the same location. Watanabe et al. [18] showed that the soil in the Kyoto area contained mutagenic compounds that might be deposits from air, and identified the major mutagens as nitrated PAHs such as 3,6-dinitrobenzo[e]pyrene. The identification of various mutagens in ambient air indicates that people inhale a mixture of various mutagens, rather than a single mutagen.
Inhalation of a mixture of mutagens has been suspected to induce DNA damage resulting in carcinogenesis in target organs and, in some cases, mutagenesis in the germ cells. Although DNA adduct formation, micronucleus induction, and DNA strand breaks in surrogate tissues, for example white blood cells, have been analyzed as biomarkers for assessing the genotoxicity of tobacco smoke [19, 20], the total mutagenicity of the environmental mixture in ambient air remains to be clarified. Formation of DNA adducts has been shown to be elevated in the white blood cells of individuals heavily exposed to air pollutants [21]; however, the amount of DNA adducts induced in target tissues, especially lung, by air-borne chemicals needs to be analyzed to assess the mutagenicity of the whole environmental mixture. Since analysis of DNA adducts in lung tissue cannot be conducted for human populations, studying the exposure of experimental animals, such as rodents, to ambient air (in situ exposure), is a limited but potentially effective method for addressing the issue of how a whole mixture of air pollutants is mutagenic [22].
Induction of somatic mutation(s) at specific sequences on proto-oncogenes and/or tumor suppressor genes is a key process in carcinogenesis. To reveal how mutation at these specific sequences is induced by environmental mutagens is an important issue for understanding the mechanism of mutagenesis and carcinogenesis induced by environment mutagens. Furthermore, mutations on the unique sequences are candidate molecular signatures for monitoring the exposure of mutagens.
Inhalation of mutagens is recognized to cause lung cancer, and air pollutants and tobacco smoke are suspected to be major causes of in vivo mutagenesis of proto-oncogenes and tumor suppressor genes in lung. Among proto-oncogenes and tumor-suppressor genes, TP53 is frequently mutated gene in lung cancer; about 40% of all lung cancer cases compiled in the IARC TP53 database [83] carry a mutated TP53 gene. A unique characteristic of TP53 mutation in lung cancer is a high rate of occurrence of G to T transversions; this rate is comparable to that of G to A transitions, which are common mutations in the TP53 gene in all types of cancer, including lung [81, 90]. The frequently mutated codons (hotspots) on the TP53 gene in lung cancer are codons 157, 158, 175, 245, 248, 249, and 273 [91].
I analyzed the IARC TP53 database to reveal the mutation spectrum at the level of nucleotide sequence of the TP53 gene in lung cancer, and potentially identify agent(s) contributing to mutagenesis of the TP53 gene. Table 1 summarizes my analysis of the base substitutions in frequently mutated codons in the TP53 gene in lung cancer [91]. It is well-known that mutations are mainly induced at CpG sites on the TP53 gene in human cancer [81]. As shown in Table 1, G to T transversions were induced in lung cancer on 5 guanine residues centered in CGN triplets at nucleotide #12457 of codon 157 (CGT to CTT), #12461 of codon 158 (CGC to CTC), #13370 of codon 245 (CGG to CTG), #13380 of codon 248 (CGG to CTG), and #13799 of codon 273 (CGT to CTT). The triplets (CGC, CGT, and CGG), in which G to T transversions were induced in the TP53 gene, were identical to those containing the BaP-induced mutation hotspots (nucleotide numbers 125, 140, 143, and 413 on the gpt gene) in the lungs of gpt delta mice [50]. These observations confirm the speculation that G to T transversions on mutated TP53 genes in lung cancer may be induced by BaP and other carcinogenic PAHs contained in tobacco smoke [90, 91, 97].
G to A transitions were also frequently induced in lung cancer. G to A transitions were induced at 4 guanine residues, that is, at nucleotide #12512 of codon 175 (CGC to CAC), #13380 and #13381 of codon 248 (CGG to CAG), and #13799 of codon 273 (CGT to CAT). Among these triplets, guanine residues centered in CGT and CGG were also mutation hotspots for G to A transitions induced by inhalation of diesel exhaust in the lungs of gpt delta mice (nucleotide numbers 64, 110, and 115 of the gpt gene) [54]. Again, these findings indicate that air pollutants emitted from diesel engines and other fossil fuel combustion processes may contribute, at least partly, to mutagenesis of the TP53 gene, but the possibility that spontaneous mutations were enhanced on frequently mutated codons on TP53 gene cannot be ruled out, because G to A transitions occur frequently as spontaneous mutations. Nevertheless, it is clear that comparison between the mutation spectra of proto-oncogenes and tumor suppressor genes in lung cancers and those of in vivo mutations in transgenic rodent assays can provide clues to the identify of environmental mutagens that cause cancer.
Because the combinations of BCL11A plus HBG-197 or BCL11A plus HBG-115 edits achieved the highest HbF levels in vitro, we pursued the in vivo evaluation of their engraftability, retention of genome editing and the HbF expression/reactivation in engrafted erythroid cells. To this aim, CD34+ cells were expanded for 2 days in cytokine-enriched medium supplemented with 3 small molecules (StemRegenin1, UM171, Ly2228820) to achieve HSC expansion before transfection as we have previously described.4,12 As controls, we used CD34+ cells that were either untransfected or transfected with any of the 3 single RNPs. Two days posttransfection, the cells were transplanted in a NBSGW mouse model without conditioning. The mice were euthanized 16 weeks posttransplantation (Figure 4A). We did not observe a difference in overall engraftment levels of human CD45+ cells in any of the assayed conditions. Multilineage engraftment was also evident in all mice, with no skewing toward a particular lineage in any of the different groups (Figure 4B). Assessment of the bone marrow HbF percentage expression within the erythroid Glycophorin A (GlyA)+ cells in transplanted mice revealed that single editing of either the BCL11A enhancer or HBG-115 could yield up to 71% F+ cells, whereas editing of the HBG-197 was be457b7860
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