Generation and characterization of a human iPSC cell line expressing inducible Cas9 in the “safe harbor” AAVS1 locus

Castaño, Julio; Bueno, Clara; Jiménez‐Delgado, Senda; Roca-Ho, Heleia; Fraga, Mario F.; Fernández, Agustín F.; Nakanishi, Mahito; Torres-Ruíz, Raúl; Rodríguez‐Perales, Sandra; Menéndez, Pablo

doi:10.1016/j.scr.2017.04.011

Cited by 28 publications

(17 citation statements)

References 4 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Previous studies generated transgenic pluripotent stem cells (iPSCs) or immune cells to treat diseases using CRISPR/Cas9-based gene-editing approach by modifying AAVS1 site. 43 , 44 In this study, for the first time, we used CRISPR/Cas9 technology to successfully insert the CAR fragment into the AAVS1 site on the genome of T cells. Compared with traditional CAR-T technology, CRISPR/Cas9-editing technology can achieve the site-specific integration of CAR elements in the genome in one step without the use of viruses.…”

Section: Discussionmentioning

confidence: 99%

Nanobody-based chimeric antigen receptor T cells designed by CRISPR/Cas9 technology for solid tumor immunotherapy

Duan

Jiang

et al. 2021

Sig Transduct Target Ther

View full text Add to dashboard Cite

Chimeric antigen receptor-based T-cell immunotherapy is a promising strategy for treatment of hematological malignant tumors; however, its efficacy towards solid cancer remains challenging. We therefore focused on developing nanobody-based CAR-T cells that treat the solid tumor. CD105 expression is upregulated on neoangiogenic endothelial and cancer cells. CD105 has been developed as a drug target. Here we show the generation of a CD105-specific nanobody, an anti-human CD105 CAR-T cells, by inserting the sequences for anti-CD105 nanobody-linked standard cassette genes into AAVS1 site using CRISPR/Cas9 technology. Co-culture with CD105+ target cells led to the activation of anti-CD105 CAR-T cells that displayed the typically activated cytotoxic T-cell characters, ability to proliferate, the production of pro-inflammatory cytokines, and the specific killing efficacy against CD105+ target cells in vitro. The in vivo treatment with anti-CD105 CAR-T cells significantly inhibited the growth of implanted CD105+ tumors, reduced tumor weight, and prolonged the survival time of tumor-bearing NOD/SCID mice. Nanobody-based CAR-T cells can therefore function as an antitumor agent in human tumor xenograft models. Our findings determined that the strategy of nanobody-based CAR-T cells engineered by CRISPR/Cas9 system has a certain potential to treat solid tumor through targeting CD105 antigen.

show abstract

Section: Discussionmentioning

confidence: 99%

Nanobody-based chimeric antigen receptor T cells designed by CRISPR/Cas9 technology for solid tumor immunotherapy

Duan

Jiang

et al. 2021

Sig Transduct Target Ther

View full text Add to dashboard Cite

show abstract

“…Precise integration of exogenous DNA into the genome is often performed by targeting a genomic ‘safe harbour’ locus that can tolerate gene insertion with few deleterious effects and limited transgene silencing. The AAVS1 locus is a popular choice for targeted knock in of exogenous DNA 16 , 17 . This region of the genome is claimed to facilitate robust and persistent transgene expression 11 , aided by flanking insulator regions 10 .…”

Section: Discussionmentioning

confidence: 99%

Variable expression and silencing of CRISPR-Cas9 targeted transgenes identifies the AAVS1 locus as not an entirely safe harbour

et al. 2020

View full text Add to dashboard Cite

Background: Diseases such as hypertrophic cardiomyopathy (HCM) can lead to severe outcomes including sudden death. The generation of human induced pluripotent stem cell (hiPSC) reporter lines can be useful for disease modelling and drug screening by providing physiologically relevant in vitro models of disease. The AAVS1 locus is cited as a safe harbour that is permissive for stable transgene expression, and hence is favoured for creating gene targeted reporter lines. Methods: We generated hiPSC reporters using a plasmid-based CRISPR/Cas9 nickase strategy. The first intron of PPP1R12C, the AAVS1 locus, was targeted with constructs expressing a genetically encoded calcium indicator (R-GECO1.0) or HOXA9-T2A-mScarlet reporter under the control of a pCAG or inducible pTRE promoter, respectively. Transgene expression was compared between clones before, during and/or after directed differentiation to mesodermal lineages. Results: Successful targeting to AAVS1 was confirmed by PCR and sequencing. Of 24 hiPSC clones targeted with pCAG-R-GECO1.0, only 20 expressed the transgene and in these, the percentage of positive cells ranged from 0% to 99.5%. Differentiation of a subset of clones produced cardiomyocytes, wherein the percentage of cells positive for R-GECO1.0 ranged from 2.1% to 93.1%. In the highest expressing R-GECO1.0 clones, transgene silencing occurred during cardiomyocyte differentiation causing a decrease in expression from 98.93% to 1.3%. In HOXA9-T2A-mScarlet hiPSC reporter lines directed towards mesoderm lineages, doxycycline induced a peak in transgene expression after two days but this reduced by up to ten-thousand-fold over the next 8-10 days. Nevertheless, for R-GECO1.0 lines differentiated into cardiomyocytes, transgene expression was rescued by continuous puromycin drug selection, which allowed the Ca2+ responses associated with HCM to be investigated in vitro using single cell analysis. Conclusions: Targeted knock-ins to AAVS1 can be used to create reporter lines but variability between clones and transgene silencing requires careful attention by researchers seeking robust reporter gene expression.

show abstract

“…Moreover, this strategy can reuse effective and safe RGNs for the addition of different transgenes to the same site, which lowers the development effort for individual therapies. Besides the AAV integration site 1 ( AAVS1 ) locus in human hematopoietic stem and progenitor cells (HSPCs) [ 60 ], the HPRT gene locus has also been employed as a safe harbor locus for TI in human cells [ 61 ], as has the chemokine (CC motif) receptor 5 ( CCR5 ) [ 62 ], and the popular murine ROSA26 locus has also found its ortholog in the human genome [ 63 ]. TI strategies that disrupt HPRT function can additionally enrich modified cells by selection against non-integrants with 6-thioguanine in vitro [ 64 ] and, with mixed results, in vivo [ 64 , 65 ].…”

Section: Rare Repair: Employing the Molecular Toolkitmentioning

confidence: 99%

Rare Opportunities: CRISPR/Cas-Based Therapy Development for Rare Genetic Diseases

2019

View full text Add to dashboard Cite

Rare diseases pose a global challenge, in that their collective impact on health systems is considerable, whereas their individually rare occurrence impedes research and development of efficient therapies. In consequence, patients and their families are often unable to find an expert for their affliction, let alone a cure. The tide is turning as pharmaceutical companies embrace gene therapy development and as serviceable tools for the repair of primary mutations separate the ability to create cures from underlying disease expertise. Whereas gene therapy by gene addition took decades to reach the clinic by incremental diseasespecific refinements of vectors and methods, gene therapy by genome editing in its basic form merely requires certainty about the causative mutation. Suddenly we move from concept to trial in 3 years instead of 30: therapy development in the fast lane, with all the positive and negative implications of the phrase. Since their first application to eukaryotic cells in 2013, the proliferation and refinement in particular of tools based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated protein (Cas) prokaryotic RNA-guided nucleases has prompted a landslide of therapy-development studies for rare diseases. An estimated thousands of orphan diseases are up for adoption, and legislative, entrepreneurial, and research initiatives may finally conspire to find many of them a good home. Here we summarize the most significant recent achievements and remaining hurdles in the application of CRISPR/Cas technology to rare diseases and take a glimpse at the exciting road ahead. Marina Kleanthous and Carsten W. Lederer contributed equally to this review.

show abstract

Generation and characterization of a human iPSC cell line expressing inducible Cas9 in the “safe harbor” AAVS1 locus

Cited by 28 publications

References 4 publications

Nanobody-based chimeric antigen receptor T cells designed by CRISPR/Cas9 technology for solid tumor immunotherapy

Nanobody-based chimeric antigen receptor T cells designed by CRISPR/Cas9 technology for solid tumor immunotherapy

Variable expression and silencing of CRISPR-Cas9 targeted transgenes identifies the AAVS1 locus as not an entirely safe harbour

Rare Opportunities: CRISPR/Cas-Based Therapy Development for Rare Genetic Diseases

Contact Info

Product

Resources

About