The unlimited proliferation capacity of embryonic stem cells (ESCs) combined with their pluripotent differentiation potential in various lineages raised great interest in both the scientific ...community and the public at large with hope for future prospects of regenerative medicine. However, since ESCs are derived from human embryos, their use is associated with significant ethical issues preventing broad studies and therapeutic applications. To get around this bottleneck, Takahashi and Yamanaka have recently achieved the conversion of adult somatic cells into ES-like cells via the forced expression of four transcription factors: Oct3/4, Sox2, Klf4 and c-Myc. This first demonstration attracted public attention and opened a new field of stem cells research with both cognitive - such as disease modeling - and therapeutic prospects. This pioneer work just received the 2012 Nobel Prize in Physiology or Medicine. Many methods have been reported since 2006, for the generation of induced pluripotent stem (iPS) cells. Most strategies currently under use are based on gene delivery via gamma-retroviral or lentiviral vectors; some experiments have also been successful using plasmids or transposons- based systems and few with adenovirus. However, most experiments involve integration in the host cell genome with an identified risk for insertional mutagenesis and oncogenic transformation. To circumvent such risks which are deemed incompatible with therapeutic prospects, significant progress has been made with transgene-free reprogramming methods based on e.g.: sendai virus or direct mRNA or protein delivery to achieve conversion of adult cells into iPS. In this review we aim to cover current knowledge relating to both delivery systems and combinations of inducing factors including chemicals which are used to generate human iPS cells. Finally, genetic instability resulting from the reprogramming process is also being considered as a safety bottleneck for future clinical translation and stem cell-therapy prospects based on iPS.
Self-inactivating (SIN) lentiviral vectors (LV) have an excellent therapeutic potential as demonstrated in preclinical studies and clinical trials. However, weaker mechanisms of insertional ...mutagenesis could still pose a significant risk in clinical applications. Taking advantage of novel in vivo genotoxicity assays, we tested a battery of LV constructs, including some with clinically relevant designs, and found that oncogene activation by promoter insertion is the most powerful mechanism of early vector-induced oncogenesis. SIN LVs disabled in their capacity to activate oncogenes by promoter insertion were less genotoxic and induced tumors by enhancer-mediated activation of oncogenes with efficiency that was proportional to the strength of the promoter used. On the other hand, when enhancer activity was reduced by using moderate promoters, oncogenesis by inactivation of tumor suppressor gene was revealed. This mechanism becomes predominant when the enhancer activity of the internal promoter is shielded by the presence of a synthetic chromatin insulator cassette. Our data provide both mechanistic insights and quantitative readouts of vector-mediated genotoxicity, allowing a relative ranking of different vectors according to these features, and inform current and future choices of vector design with increasing biosafety.
Retroviral vectors have induced subtle clonal skewing in many gene therapy patients and severe clonal proliferation and leukemia in some of them, emphasizing the need for comprehensive integration ...site analyses to assess the biosafety and genomic pharmacokinetics of vectors and clonal fate of gene-modified cells in vivo. Integration site analyses such as linear amplification-mediated PCR (LAM-PCR) require a restriction digest generating unevenly small fragments of the genome. Here we show that each restriction motif allows for identification of only a fraction of all genomic integrants, hampering the understanding and prediction of biological consequences after vector insertion. We developed a model to define genomic access to the viral integration site that provides optimal restriction motif combinations and minimizes the percentage of nonaccessible insertion loci. We introduce a new nonrestrictive LAM-PCR approach that has superior capabilities for comprehensive unbiased integration site retrieval in preclinical and clinical samples independent of restriction motifs and amplification inefficiency.
The main objective of oncogenetics is to characterize a subpopulation of patients at high risk of cancer development at an early age in order to provide specific recommendations for an optimized ...follow-up and care path. Oncogenetic counselling helps to assess individual risk from a family history. By a family approach of formal genetics, the key issue is to identify families with a strong aggregation of cancers, and, in particular, suggesting a specific syndrome of inherited predisposition to cancer. This approach can lead to the proposal of germline genetic testing in search of causal mutations. As up to know, the search for a constitutional mutation in the BRCA genes has led to the identification of a causal deleterious mutation in less than 10% of index-cases analyzed. It is therefore important to evaluate the impact of new genes in the current panorama of inherited predisposition to breast and ovarian cancer.
L’oncogénétique a pour objectif principal de caractériser une sous-population à haut risque de développement de cancers à un âge précoce afin de préconiser les recommandations pour un parcours ...optimisé de suivi et de soins. La consultation d’oncogénétique contribue à évaluer un risque individuel à partir d’une histoire familiale. Par une approche familiale de génétique formelle, il s’agit de repérer les familles avec une forte agrégation de cancers, éventuellement évocatrice d’un syndrome de prédisposition héréditaire. Cette démarche peut conduire à la proposition d’un test génétique constitutionnel à la recherche de mutations causales. Jusqu’à une période récente, la recherche de mutation constitutionnelle sur les gènes
BRCA
a abouti à l’identification d’une mutation délétère chez moins de 10 % des cas-index analysés. Il est donc important d’évaluer l’impact de nouveaux gènes dans le panorama actuel de la prédisposition héréditaire au cancer du sein et de l’ovaire.
The main objective of oncogenetics is to characterize a subpopulation of patients at high risk of cancer development at an early age in order to provide specific recommendations for an optimized follow-up and care path. Oncogenetic counselling helps to assess individual risk from a family history. By a family approach of formal genetics, the key issue is to identify families with a strong aggregation of cancers, and, in particular, suggesting a specific syndrome of inherited predisposition to cancer. This approach can lead to the proposal of germline genetic testing in search of causal mutations. As up to know, the search for a constitutional mutation in the BRCA genes has led to the identification of a causal deleterious mutation in less than 10% of index-cases analyzed. It is therefore important to evaluate the impact of new genes in the current panorama of inherited predisposition to breast and ovarian cancer.
Les progrès du séquençage à haut débit permettent de rechercher simultanément des mutations sur plusieurs gènes pour explorer la prédisposition héréditaire au cancer du sein. Selon le gène, le niveau ...de risque et le spectre des cancers peuvent varier. Les dispositions spécifiques de prise en charge préconisées sont modulées en fonction des gènes, classés en : (1) très haut risque, tels les gènes BRCA1/2 suivant les recommandations de l’INCa 2017 ; (2) risque élevé ; (3) augmentation modérée : dans ce dernier cas, les mesures de surveillance sont similaires à la population générale. En l’absence de mutation, d’autres facteurs de risque peuvent intervenir et des scores professionnels être calculés. Cependant, selon les recommandations de la HAS 2014, l’histoire familiale prévaut : sur cette base, le dispositif national d’oncogénétique de l’INCa a mis en place un maillage national de réseaux de suivi des personnes à haut risque, présentant ou non des mutations. Enfin, de nouvelles voies thérapeutiques spécifiques s’ouvrent pour les personnes porteuses de mutations.
Recent progres in massive deep sequencing have paved the way to routine exploration of the current panorama of inherited predisposition to breast cancer, through multigene panel analysis. Cancer risks and spectrum vary according to the gene at stake. Surveillance modalities are thus adapted to the risk-level : (1) very high, which follow specific recommendations from french NCI (INCa) ; (2) high and (3) moderate ; in the latter case, patients’follow-up compares to women of the general population. In the absence of causal mutation, other risk factors may be considered and professional scores be calculated. Of note and according to french HAS recommendations issued in 2014, the family history prevails ; on this basis, INCa has set up a national coverage of follow-up networks which specifically address high risk individuals, with or without mutation. Finally, an innovative path of specific targeted-therapies is currently opening for mutation carriers.
Les progrès du séquençage à haut débit permettent de rechercher simultanément des mutations sur plusieurs gènes pour explorer la prédisposition héréditaire au cancer du sein. Selon le gène, le niveau ...de risque et le spectre des cancers peuvent varier. Les dispositions spécifiques de prise en charge préconisées sont modulées en fonction des gènes, classés en : (1) très haut risque, tels les gènes
BRCA1/2
suivant les recommandations de l’INCa 2017 ; (2) risque élevé ; (3) augmentation modérée : dans ce dernier cas, les mesures de surveillance sont similaires à la population générale. En l’absence de mutation, d’autres facteurs de risque peuvent intervenir et des scores professionnels être calculés. Cependant, selon les recommandations de la HAS 2014, l’histoire familiale prévaut : sur cette base, le dispositif national d’oncogénétique de l’INCa a mis en place un maillage national de réseaux de suivi des personnes à haut risque, présentant ou non des mutations. Enfin, de nouvelles voies thérapeutiques spécifiques s’ouvrent pour les personnes porteuses de mutations.
Recent progres in massive deep sequencing have paved the way to routine exploration of the current panorama of inherited predisposition to breast cancer, through multigene panel analysis. Cancer risks and spectrum vary according to the gene at stake. Surveillance modalities are thus adapted to the risk-level : (1) very high, which follow specific recommendations from french NCI (INCa) ; (2) high and (3) moderate ; in the latter case, patients’follow-up compares to women of the general population. In the absence of causal mutation, other risk factors may be considered and professional scores be calculated. Of note and according to french HAS recommendations issued in 2014, the family history prevails ; on this basis, INCa has set up a national coverage of follow-up networks which specifically address high risk individuals, with or without mutation. Finally, an innovative path of specific targeted-therapies is currently opening for mutation carriers.
L’oncogénétique a pour objectif principal de caractériser une sous-population à haut risque de développement de cancers à un âge précoce afin de préconiser les recommandations pour un parcours ...optimisé de suivi et de soins. La consultation d’oncogénétique contribue à évaluer un risque individuel à partir d’une histoire familiale. Par une approche familiale de génétique formelle, il s’agit de repérer les familles avec une forte agrégation de cancers, éventuellement évocatrice d’un syndrome de prédisposition héréditaire. Cette démarche peut conduire à la proposition d’un test génétique constitutionnel à la recherche de mutations causales. Jusqu’à une période récente, la recherche de mutation constitutionnelle sur les gènes BRCA a abouti à l’identification d’une mutation délétère chez moins de 10 % des cas-index analysés. Il est donc important d’évaluer l’impact de nouveaux gènes dans le panorama actuel de la prédisposition héréditaire au cancer du sein et de l’ovaire.
The main objective of oncogenetics is to characterize a subpopulation of patients at high risk of cancer development at an early age in order to provide specific recommendations for an optimized follow-up and care path. Oncogenetic counselling helps to assess individual risk from a family history. By a family approach of formal genetics, the key issue is to identify families with a strong aggregation of cancers, and, in particular, suggesting a specific syndrome of inherited predisposition to cancer. This approach can lead to the proposal of germline genetic testing in search of causal mutations. As up to know, the search for a constitutional mutation in the BRCA genes has led to the identification of a causal deleterious mutation in less than 10% of index-cases analyzed. It is therefore important to evaluate the impact of new genes in the current panorama of inherited predisposition to breast and ovarian cancer.
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