Search Results (20)

Mucopolysaccharidosis (MPS) IVA is a bone-affecting lysosomal storage disease (LSD) caused by impaired degradation of the glycosaminoglycans (GAGs) keratan sulfate (KS) and chondroitin 6-sulfate (C6S) due to deficient N-acetylgalactosamine-6-sulfatase (GALNS) enzyme activity. Previously, we successfully developed and validated a CRISPR/nCas9-based gene therapy (GT) to insert an expression cassette at the AAVS1 and ROSA26 loci in human MPS IVA fibroblasts and MPS IVA mice, respectively. In this study, we have extended our approach to evaluate the effectiveness of our CRISPR/nCas9-based GT in editing human CD34+ cells to mediate cross-correction of MPS IVA fibroblasts. CD34+ cells were electroporated with the CRISPR/nCas9 system, targeting the AAVS1 locus. The nCas9-mediated on-target donor template insertion, and the stemness of the CRISPR/nCas-edited CD34+ cells was evaluated. Additionally, MPS IVA fibroblasts were co-cultured with CRISPR/nCas-edited CD34+ cells to assess cross-correction. CRISPR/nCas9-based gene editing did not affect the stemness of CD34+ cells but did lead to supraphysiological levels of the GALNS enzyme. Upon co-culture, MPS IVA fibroblasts displayed a significant increase in the GALNS enzyme activity along with lysosomal mass reduction, pro-oxidant profile amelioration, mitochondrial mass recovery, and pro-apoptotic and pro-inflammatory profile improvement. These results show the potential of our CRISPR/nCas9-based GT to edit CD34+ cells to mediate cross-correction. Full article

Background: Tauopathy has been identified as a prevalent causative agent of neurodegenerative diseases, including frontotemporal dementia with parkinsonism-17 (FTDP-17). This rare hereditary neurodegenerative condition is characterised by the manifestation of parkinsonism and behavioural changes. The majority of cases of FTDP-17 are associated with mutations in the MAPT gene, which encodes the tau protein. MAPT mutations lead to disruption of the balance between 3R and 4R tau forms, which causes destabilisation of microtubules and impairment of cellular organelle functions, particularly mitochondrial dysfunction. The development of model systems and tools for studying the molecular, genetic, and biochemical mechanisms underlying FTDP-17 and testing therapies at the cellular level is an urgent necessity. Methods: In this study, we generated transgenic lines of induced pluripotent stem cells (iPSCs) from a patient carrying the pathogenic mutation c.2013T > G (rs63750756, p.N279K) of MAPT and a healthy donor. A doxycycline-controlled transgene of the genetically encoded biosensor MitoTimer was integrated into the AAVS1 locus of these cells. The MitoTimer biosensor allows for lifetime monitoring of the turnover of mitochondria in neuronal cells derived from directed iPSC differentiation. The fact that transcription of the transgene can be induced by doxycycline provides additional possibilities for pulse labelling of newly formed mitochondria. Results: Transgenic iPSC lines provide a unique tool to study the molecular and genetic mechanisms of FTDP-17 caused by the presence of the c.2013T > G (p.N279K) mutation, as well as to test potential drugs in vitro. Full article

The CRISPR/Cas9 system enables precise and efficient modification of eukaryotic genomes. Among its various applications, homology-directed repair (HDR) mediated knock-in (KI) is crucial for creating human disease models, gene therapy, and agricultural genetic enhancements. Despite its potential, HDR-mediated knock-in efficiency remains relatively low. This study investigated the impact of 5′ end PEG10 modification on site-specific integration of the target gene. The HEK293 cell line is considered a highly attractive expression system for the production of recombinant proteins, with the construction of site-specific integration cell lines at the AAVS1 locus enabling stable protein expression. This study investigated the impact of the 5′ end PEG10 modification on the site-specific integration of the target gene at the AAVS1 locus in the 293T cell line. Utilizing this 5′ end PEG10 modification resulted in a 1.9-fold increase in knock-in efficiency for a 1.8 kb target fragment, improving efficiency from 26% to 49%. An optimized system was utilized to successfully establish a high-expression, site-specific integration 293T cell line for TAT-Cas9-EGFP, providing a reliable resource of seed cells for subsequent protein production. Full article

Dopaminergic neurons are the predominant brain cells affected in Parkinson’s disease. With the limited availability of live human brain dopaminergic neurons to study pathological mechanisms of Parkinson’s disease, dopaminergic neurons have been generated from human-skin-cell-derived induced pluripotent stem cells. Originally, induced pluripotent stem-cell-derived dopaminergic neurons were generated using small molecules. These neurons took more than two months to mature. However, the transcription-factor-mediated differentiation of induced pluripotent stem cells has revealed quicker and cheaper methods to generate dopaminergic neurons. In this study, we compared and contrasted three protocols to generate induced pluripotent stem-cell-derived dopaminergic neurons using transcription-factor-mediated directed differentiation. We deviated from the established protocols using lentivirus transduction to stably integrate different transcription factors into the AAVS1 safe harbour locus of induced pluripotent stem cells. We used different media compositions to generate more than 90% of neurons in the culture, out of which more than 85% of the neurons were dopaminergic neurons within three weeks. Therefore, from our comparative study, we reveal that a combination of transcription factors along with small molecule treatment may be required to generate a pure population of human dopaminergic neurons. Full article

Endoplasmic reticulum (ER) stress is involved in the pathogenesis of many human diseases, such as cancer, type 2 diabetes, kidney disease, atherosclerosis and neurodegenerative diseases, in particular Parkinson’s disease (PD). Since there is currently no treatment for PD, a better understanding of the molecular mechanisms underlying its pathogenesis, including the mechanisms of the switch from adaptation in the form of unfolded protein response (UPR) to apoptosis under ER stress conditions, may help in the search for treatment methods. Genetically encoded biosensors based on fluorescent proteins are suitable tools that facilitate the study of living cells and visualization of molecular events in real time. The combination of technologies to generate patient-specific iPSC lines and genetically encoded biosensors allows the creation of cell models with new properties. Using CRISPR-Cas9-mediated homologous recombination at the AAVS1 locus of iPSC with the genetic variant p.N370S (rs76763715) in the GBA1 gene, we created a cell model designed to study the activation conditions of the IRE1-XBP1 cascade of the UPR system. The cell lines obtained have a doxycycline-dependent expression of the genetically encoded biosensor XBP1-TagRFP, possess all the properties of human pluripotent cells, and can be used to test physical conditions and chemical compounds that affect the development of ER stress, the functioning of the UPR system, and in particular, the IRE1-XBP1 cascade. Full article

Advances in stem cell technologies open up new avenues for modelling development and diseases. The success of these pursuits, however, relies on the use of cells most relevant to those targeted by the disease of interest, for example, midbrain dopaminergic neurons for Parkinson’s disease. In the present study, we report the generation of a human induced pluripotent stem cell (iPSC) line capable of purifying and tracing nascent midbrain dopaminergic progenitors and their differentiated progeny via the expression of a Blue Fluorescent Protein (BFP). This was achieved by CRISPR/Cas9-assisted knock-in of BFP and Cre into the safe harbour locus AAVS1 and an early midbrain dopaminergic lineage marker gene LMX1A, respectively. Immunocytochemical analysis and single-cell RNA sequencing of iPSC-derived neural cultures confirm developmental recapitulation of the human fetal midbrain and high-quality midbrain cells. By modelling Parkinson’s disease-related drug toxicity using 1-Methyl-4-phenylpyridinium (MPP⁺), we showed a preferential reduction of BFP⁺ cells, a finding demonstrated independently by cell death assays and single-cell transcriptomic analysis of MPP⁺ treated neural cultures. Together, these results highlight the importance of disease-relevant cell types in stem cell modelling. Full article

CAR-T cell therapy involves genetically engineering T cells to recognize and attack tumour cells by adding a chimeric antigen receptor (CAR) to their surface. In this study, we have used dual transduction with AAV serotype 6 (AAV6) to integrate an anti-CD19 CAR into human T cells at a known genomic location. The first viral vector expresses the Cas9 endonuclease and a guide RNA (gRNA) targeting the T cell receptor alpha constant locus, while the second vector carries the DNA template for homology-mediated CAR insertion. We evaluated three gRNA candidates and determined their efficiency in generating indels. The AAV6 successfully delivered the CRISPR/Cas9 machinery in vitro, and molecular analysis of the dual transduction showed the integration of the CAR transgene into the desired location. In contrast to the random integration methods typically used to generate CAR-T cells, targeted integration into a known genomic locus can potentially lower the risk of insertional mutagenesis and provide more stable levels of CAR expression. Critically, this method also results in the knockout of the endogenous T cell receptor, allowing target cells to be derived from allogeneic donors. This raises the exciting possibility of “off-the-shelf” universal immunotherapies that would greatly simplify the production and administration of CAR-T cells. Full article

Pluripotent stem cells (PSCs) offer an exciting resource for probing human biology; however, gene-editing efficiency remains relatively low in many cell types, including stem cells. Gene-editing using the CRISPR-Cas9 system offers an attractive solution that improves upon previous gene-editing approaches; however, like other technologies, off-target mutagenesis remains a concern. High-fidelity Cas9 variants greatly reduce off-target mutagenesis and offer a solution to this problem. To evaluate their utility as part of a cell-based gene-editing platform, human PSC lines were generated with a high-fidelity (HF) tetracycline-inducible engineered Streptococcus pyogenes SpCas9 (HF-iCas9) integrated into the AAVS1 safe harbor locus. By engineering cells with controllable expression of Cas9, we eliminated the need to include a large Cas9-expressing plasmid during cell transfection. Delivery of genetic cargo was further optimized by packaging DNA targeting guide RNAs (gRNAs) and donor fragments into a single plasmid backbone. The potential of homology-directed repair (HDR) based gene knock-in at the CLYBL safe harbor site and endogenous SOX2 and SIX6 genes were demonstrated. Moreover, we used non-homologous end-joining (NHEJ) for gene knockout of disease-relevant alleles. These high-fidelity CRISPR tools and the resulting HF-iCas9 cell lines will facilitate the production of cell-type reporters and mutants across different genetic backgrounds. Full article

Persistent and efficient therapeutic protein expression in the specific target cell is a significant concern in gene therapy. The controllable integration site, suitable promoter, and proper codon usage influence the effectiveness of the therapeutic outcome. Previously, we developed a non-immunoglobulin scaffold, alpha repeat protein (αRep4E3), as an HIV-1 RNA packaging interference system in SupT1 cells using the lentiviral gene transfer. Although the success of anti-HIV-1 activity was evidenced, the integration site is uncontrollable and may not be practical for clinical translation. In this study, we use the CRISPR/Cas9 gene editing technology to precisely knock-in αRep4E3 genes into the adeno-associated virus integration site 1 (AAVS1) safe harbor locus of the target cells. We compare the αRep4E3 expression under the regulation of three different promoters, including cytomegalovirus (CMV), human elongation factor-1 alpha (EF1α), and ubiquitin C (UbC) promoters with and without codon optimization in HEK293T cells. The results demonstrated that the EF1α promoter with codon-optimized αRep4E3mCherry showed higher protein expression than other promoters with non-optimized codons. We then performed a proof-of-concept study by knocking in the αRep4E3mCherry gene at the AAVS1 locus of the Jurkat cells. The results showed that the αRep4E3mCherry-expressing Jurkat cells exhibited anti-HIV-1 activities against HIV-1NL4-3 strain as evidenced by decreased capsid (p24) protein levels and viral genome copies as compared to the untransfected Jurkat control cells. Altogether, our study demonstrates that the αRep4E3 could interfere with the viral RNA packaging and suggests that the αRep4E3 scaffold protein could be a promising anti-viral molecule that offers a functional cure for people living with HIV-1. Full article

Adeno-associated virus (AAV) vector is an efficient viral-based gene delivery tool used with many types of cells and tissues, including neuronal cells and muscles. AAV serotype 6 (AAV-6), one of numerous AAV serotypes, was recently found to efficiently transduce mouse preimplantation embryos. Furthermore, through coupling with a clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system—a modern genome editing technology—AAV-6 has been shown to effectively create a mutation at a target locus, which relies on isolation of zygotes, in vitro viral infection, and transplantation of the infected embryos to recipient females. Unfortunately, this procedure, termed “ex vivo handling of embryos”, requires considerable investment of capital, time, and effort. Direct transduction of preimplantation embryos through the introduction of AAV-6 into the oviductal lumen of pregnant females would be an ideal approach. In this study, we injected various types of recombinant AAV vectors (namely, rAAV-CAG-EGFP-1, -2, -5, and -6, each carrying an enhanced green fluorescent protein [EGFP] cDNA whose expression is under the influence of a cytomegalovirus enhancer + chicken β-actin promoter) into the ampulla region of oviducts in pregnant female mice at Day 0.7 of pregnancy (corresponding to the late 1-cell stage), and EGFP-derived green fluorescence was assessed in the respective morulae. The highest levels of fluorescence were observed in rAAV-CAG-EGFP-6. The oviductal epithelium was distinctly fluorescent. The fluorescence in embryos peaked at the morula stage. Our results indicate that intra-oviductal injection of AAV-6 vectors is the most effective method for transducing zona pellucida-enclosed preimplantation embryos in situ. AAV-6 vectors could be a useful tool in the genetic manipulation of early embryos, as well as oviductal epithelial cells. Full article

Application of the CRISPR/Cas9 system to knock in fluorescent proteins to endogenous genes of interest in human pluripotent stem cells (hPSCs) has the potential to facilitate hPSC-based disease modeling, drug screening, and optimization of transplantation therapy. To evaluate the capability of fluorescent reporter hPSC lines for high-content screening approaches, we targeted EGFP to the endogenous OCT4 locus. Resulting hPSC–OCT4–EGFP lines generated expressed EGFP coincident with pluripotency markers and could be adapted to multi-well formats for high-content screening (HCS) campaigns. However, after long-term culture, hPSCs transiently lost their EGFP expression. Alternatively, through EGFP knock-in to the AAVS1 locus, we established a stable and consistent EGFP-expressing hPSC–AAVS1–EGFP line that maintained EGFP expression during in vitro hematopoietic and neural differentiation. Thus, hPSC–AAVS1–EGFP-derived sensory neurons could be adapted to a high-content screening platform that can be applied to high-throughput small-molecule screening and drug discovery campaigns. Our observations are consistent with recent findings indicating that high-frequency on-target complexities appear following CRISPR/Cas9 genome editing at the OCT4 locus. In contrast, we demonstrate that the AAVS1 locus is a safe genomic location in hPSCs with high gene expression that does not impact hPSC quality and differentiation. Our findings suggest that the CRISPR/Cas9-integrated AAVS1 system should be applied for generating stable reporter hPSC lines for long-term HCS approaches, and they underscore the importance of careful evaluation and selection of the applied reporter cell lines for HCS purposes. Full article

Induced pluripotent stem cell (iPSC)-derived cell products hold great promise as a potential cell source in personalized medicine. As concerns about the potential risk of graft-related severe adverse events, such as tumor formation from residual pluripotent cells, currently restrict their applicability, we established an optimized tool for therapeutic intervention that allows drug-controlled, specific and selective ablation of either iPSCs or the whole graft through genetic safety switches. To identify the best working system, different tools for genetic iPSC modification, promoters to express safety switches and different safety switches were combined. Suicide effects were slightly stronger when the suicide gene was delivered through lentiviral (LV) vectors compared to integration into the AAVS1 locus through TALEN technology. An optimized HSV-thymidine kinase and the inducible Caspase 9 both mediated drug-induced, efficient in vitro elimination of transgene-positive iPSCs. Choice of promoter allowed selective elimination of distinct populations within the graft: the hOct4 short response element restricted transgene expression to iPSCs, while the CAGs promoter ubiquitously drove expression in iPSCs and their progeny. Remarkably, both safety switches were able to prevent in vivo teratoma development and even effectively eliminated established teratomas formed by LV CAGs-transgenic iPSCs. These optimized tools to increase safety provide an important step towards clinical application of iPSC-derived transplants. Full article

Patient material from rare diseases such as very early-onset inflammatory bowel disease (VEO-IBD) is often limited. The use of patient-derived induced pluripotent stem cells (iPSCs) for disease modeling is a promising approach to investigate disease pathomechanisms and therapeutic strategies. We successfully developed VEO-IBD patient-derived iPSC lines harboring a mutation in the IL-10 receptor β-chain (IL-10RB) associated with defective IL-10 signaling. To characterize the disease phenotype, healthy control and VEO-IBD iPSCs were differentiated into macrophages. IL-10 stimulation induced characteristic signal transducer and activator of transcription 3 (STAT3) and suppressor of cytokine signaling 3 (SOCS3) downstream signaling and anti-inflammatory regulation of lipopolysaccharide (LPS)-mediated cytokine secretion in healthy control iPSC-derived macrophages. In contrast, IL-10 stimulation of macrophages derived from patient iPSCs did not result in STAT3 phosphorylation and subsequent SOCS3 expression, recapitulating the phenotype of cells from patients with IL-10RB deficiency. In line with this, LPS-induced cytokine secretion (e.g., IL-6 and tumor necrosis factor-α (TNF-α)) could not be downregulated by exogenous IL-10 stimulation in VEO-IBD iPSC-derived macrophages. Correction of the IL-10RB defect via lentiviral gene therapy or genome editing in the adeno-associated virus integration site 1 (AAVS1) safe harbor locus led to reconstitution of the anti-inflammatory response. Corrected cells showed IL-10RB expression, IL-10-inducible phosphorylation of STAT3, and subsequent SOCS3 expression. Furthermore, LPS-mediated TNF-α secretion could be modulated by IL-10 stimulation in gene-edited VEO-IBD iPSC-derived macrophages. Our established disease models provide the opportunity to identify and validate new curative molecular therapies and to investigate phenotypes and consequences of additional individual IL-10 signaling pathway-dependent VEO-IBD mutations. Full article

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)-mediated genome editing holds remarkable promise for the treatment of human genetic diseases. However, the possibility of off-target Cas9 activity remains a concern. To address this issue using clinically relevant target cells, we electroporated Cas9 ribonucleoprotein (RNP) complexes (independently targeted to two different genomic loci, the CXCR4 locus on chromosome 2 and the AAVS1 locus on chromosome 19) into human mobilized peripheral blood-derived hematopoietic stem and progenitor cells (HSPCs) and assessed the acquisition of somatic mutations in an unbiased, genome-wide manner via whole genome sequencing (WGS) of single-cell-derived HSPC clones. Bioinformatic analysis identified >20,000 total somatic variants (indels, single nucleotide variants, and structural variants) distributed among Cas9-treated and non-Cas9-treated control HSPC clones. Statistical analysis revealed no significant difference in the number of novel non-targeted indels among the samples. Moreover, data analysis showed no evidence of Cas9-mediated indel formation at 623 predicted off-target sites. The median number of novel single nucleotide variants was slightly elevated in Cas9 RNP-recipient sample groups compared to baseline, but did not reach statistical significance. Structural variants were rare and demonstrated no clear causal connection to Cas9-mediated gene editing procedures. We find that the collective somatic mutational burden observed within Cas9 RNP-edited human HSPC clones is indistinguishable from naturally occurring levels of background genetic heterogeneity. Full article

We have recently developed surface oligopeptide knock-in for rapid target selection (SORTS), a novel method to isolate mammalian cells with gene modifications using FACS-sorting. It relies on CRISPR/Cas9-mediated targeted knock-in of a very short promoterless expression construct (250 bp) comprising a Flag or HA epitope embedded into the smallest GPI-protein CD52, and a polyA signal from the beta-globin. CD52 efficiently delivers the epitope to the cell surface, where it serves as a marker for selection, while polyA terminates transcription and silences target gene expression. Primarily, SORTS was developed to knock out genes encoding intracellular or secreted proteins, which cannot be used as markers for selection of live cells. Using in-frame modification of SORTS, we demonstrated the possibility of HIV-1 provirus inactivation via sorting of GPI-tag positive cells. In order to make the “cured” cells resistant to a subsequent HIV-1 infection, the epitope tag in the CD52 molecule was substituted by one of the fusion inhibitor peptides from the CHR-domain of gp41. We selected a series of cell-surface-expressed, GPI-anchored, C34-based peptides that confer a strong cellular resistance to HIV-1 infection mediated by NL4-3, JRFL, or ZM153 Env. These findings together with a monoclonal antibody raised against the C34 peptide provide an opportunity to generate and select HIV-resistant lymphocytes for a therapeutic goal. SORTS was also adapted to engineer transgenic HIV-1 effector Т cells and to study cell-to-cell transmission. To facilitate transgenesis, we developed a knock-in strategy to express GPI-tag from the intronic region of the human PPP1R12C gene (AAVS1 locus) and delivered FRT sites of recombination into both alleles. In summary, SORTS is a novel instrument to isolate rare cells with precise genomic modifications with broad applications, including HIV biology. This work was supported by the Russian Science Foundation (grant 18-14-00333) and the Russian Foundation for Basic Research (grants 18-29-07052, 18-04-01016). Full article