Precise in vivo genome editing via single homology arm donor mediated intron-targeting gene integration for genetic disease correction

In vivo genome editing represents a powerful strategy for both understanding basic biology and treating inherited diseases. However, it remains a challenge to develop universal and efficient in vivo genome-editing tools for tissues that comprise diverse cell types in either a dividing or non-dividing state. Here, we describe a versatile in vivo gene knock-in methodology that enables the targeting of a broad range of mutations and cell types through the insertion of a minigene at an intron of the target gene locus using an intracellularly linearized single homology arm donor. As a proof-of-concept, we focused on a mouse model of premature-aging caused by a dominant point mutation, which is difficult to repair using existing in vivo genome-editing tools. Systemic treatment using our new method ameliorated aging-associated phenotypes and extended animal lifespan, thus highlighting the potential of this methodology for a broad range of in vivo genome-editing applications

Modelling Fanconi anemia pathogenesis and therapeutics using integration-free patient-derived iPSCs.

Fanconi anaemia (FA) is a recessive disorder characterized by genomic instability, congenital abnormalities, cancer predisposition and bone marrow (BM) failure. However, the pathogenesis of FA is not fully understood partly due to the limitations of current disease models. Here, we derive integration free-induced pluripotent stem cells (iPSCs) from an FA patient without genetic complementation and report in situ gene correction in FA-iPSCs as well as the generation of isogenic FANCA-deficient human embryonic stem cell (ESC) lines. FA cellular phenotypes are recapitulated in iPSCs/ESCs and their adult stem/progenitor cell derivatives. By using isogenic pathogenic mutation-free controls as well as cellular and genomic tools, our model serves to facilitate the discovery of novel disease features. We validate our model as a drug-screening platform by identifying several compounds that improve hematopoietic differentiation of FA-iPSCs. These compounds are also able to rescue the hematopoietic phenotype of FA patient BM cells

Targeted gene correction of laminopathy-associated LMNA mutations in patient-specific iPSCs

International audienceCombination of stem cell-based approaches with gene-editing technologies represents an attractive strategy for studying human disease and developing therapies. However, gene-editing methodologies described to date for human cells suffer from technical limitations including limited target gene size, low targeting efficiency at transcriptionally inactive loci, and off-target genetic effects that could hamper broad clinical application. To address these limitations, and as a proof of principle, we focused on homologous recombination-based gene correction of multiple mutations on lamin A (LMNA), which are associated with various degenerative diseases. We show that helper-dependent adenoviral vectors (HDAdVs) provide a highly efficient and safe method for correcting mutations in large genomic regions in human induced pluripotent stem cells and can also be effective in adult human mesenchymal stem cells. This type of approach could be used to generate genotype-matched cell lines for disease modeling and drug discovery and potentially also in therapeutics

Modeling Fanconi anemia pathogenesis and therapeutics using integration-free patient iPSCs

Fanconi anaemia (FA) is a recessive disorder characterized by genomic instability, congenital abnormalities, cancer predisposition and bone marrow (BM) failure. However, the pathogenesis of FA is not fully understood partly due to the limitations of current disease models. Here, we derive integration free-induced pluripotent stem cells (iPSCs) from an FA patient without genetic complementation and report in situ gene correction in FA-iPSCs as well as the generation of isogenic FANCA-deficient human embryonic stem cell (ESC) lines. FA cellular phenotypes are recapitulated in iPSCs/ESCs and their adult stem/progenitor cell derivatives. By using isogenic pathogenic mutation-free controls as well as cellular and genomic tools, our model serves to facilitate the discovery of novel disease features. We validate our model as a drug-screening platform by identifying several compounds that improve hematopoietic differentiation of FA-iPSCs. These compounds are also able to rescue the hematopoietic phenotype of FA patient BM cells.Altres ajuts: Strategic Priority Research Program of the Chinese Academy of Sciences (XDA01020312), National Basic Research Program of China (973 Program,2014CB964600;2014CB910500), NSFC (81271266, 31222039, 81330008, 31201111, 81371342, 81300261, 81300677), Key Research Program of the Chinese Academy of Sciences (KJZD-EW-TZ-L05), Beijing Natural Science Foundation (7141005; 5142016), the Thousand Young Talents program of China, National Laboratory of Biomacromolecules (012kf02, 2013kf05;2013kf11;2014kf02), and State Key Laboratory of Drug Research (SIMM1302KF-17). M.L. and K.S. are supported by CIRM fellowship. N.M was partially supported by La Fundació Privada La Marató de TV3, 121430/31/32. Y.T. was partially supported by an Uehara Memorial Foundation research fellowship. E.N. was partially supported by an F.M. Kirby Foundation postdoctoral fellowship. J.S. was supported by Fundació Marató TV3 (464/C/2012). J.A.B. was supported by grants from La Fundació Privada La Marató de TV3, 121430/31/32. J.C.I.B. was supported by grants from the G. Harold and Leila Y. Mathers Charitable Foundation, The California Institute of Regenerative Medicine, Ellison Medical Foundation, and The Leona M. and Harry B. Helmsley Charitable Trust grant #2012-PG-MED002

Progressive degeneration of human neural stem cells caused by pathogenic LRRK2

Nuclear architecture defects have been shown to correlate with the manifestation of a number of human diseases as well as aging(1-4). It is then plausible that diseases whose manifestations correlate with aging might be connected to the appearance of nuclear aberrations over time. We decided to evaluate nuclear organization in the context of aging-associated disorders by focusing on a Leucine Rich Repeat Kinase 2 (LRRK2) dominant mutation (G2019S) shown to associate with familial and sporadic Parkinson’s Disease (PD), as well as impairment of adult neurogenesis in mice(5). Here, we report on the generation of PD patient-derived induced pluripotent stem cells (iPSCs) and the implications of LRRK2(G2019S) in human neural stem cell (NSC) populations. Mutant NSCs showed increased susceptibility to proteasomal stress as well as passage-dependent deficiencies in clonal expansion and neuronal differentiation. Disease phenotypes were rescued by targeted correction of the LRRK2(G2019S) mutation with its wild-type counterpart in PD-iPSCs and recapitulated upon targeted knock-in of LRRK2(G2019S) in human embryonic stem cells (hESCs). Analysis of human brain tissue showed nuclear envelope impairment in clinically diagnosed Parkinson’s patients. Altogether, our results identify the nucleus as a previously unknown cellular organelle in Parkinson’s pathology and may help open new avenues for PD diagnoses as well as potential development of therapeutics targeting this fundamental cell structure

Precise in vivo genome editing via single homology arm donor mediated intron-targeting gene integration for genetic disease correction

In vivo genome editing represents a powerful strategy for both understanding basic biology and treating inherited diseases. However, it remains a challenge to develop universal and efficient in vivo genome-editing tools for tissues that comprise diverse cell types in either a dividing or non-dividing state. Here, we describe a versatile in vivo gene knock-in methodology that enables the targeting of a broad range of mutations and cell types through the insertion of a minigene at an intron of the target gene locus using an intracellularly linearized single homology arm donor. As a proof-of-concept, we focused on a mouse model of premature-aging caused by a dominant point mutation, which is difficult to repair using existing in vivo genome-editing tools. Systemic treatment using our new method ameliorated aging-associated phenotypes and extended animal lifespan, thus highlighting the potential of this methodology for a broad range of in vivo genome-editing applications