Spinal muscular atrophy (SMA) is a kind of neuromuscular disease characterized by progressive motor neuron loss in the spinal cord. It is caused by mutations in the survival motor neuron 1 (SMN1) gene. SMN1 has a paralogous gene, survival motor neuron 2 (SMN2), in humans that is present in almost all SMA patients. The generation and genetic correction of SMA patient-specific induced pluripotent stem cells (iPSCs) is a viable, autologous therapeutic strategy for the disease. Here, c-Myc-free and non-integrating iPSCs were generated from the urine cells of an SMA patient using an episomal iPSC reprogramming vector, and a unique crRNA was designed that does not have similar sequences (≤3 mismatches) anywhere in the human reference genome. In situ gene conversion of the SMN2 gene to an SMN1-like gene in SMA-iPSCs was achieved using CRISPR/Cpf1 and single-stranded oligodeoxynucleotide with a high efficiency of 4/36. Seamlessly gene-converted iPSC lines contained no exogenous sequences and retained a normal karyotype. Significantly, the SMN expression and gems localization were rescued in the gene-converted iPSCs and their derived motor neurons. This is the first report of an efficient gene conversion mediated by Cpf1 homology-directed repair in human cells and may provide a universal gene therapeutic approach for most SMA patients.

Induced pluripotent stem cells (iPSCs) are a promising source of mesenchymal stem cells (MSCs) for clinical applications. In this study, we transformed human iPSCs using a non-viral vector carrying the IL24 transgene pHrn-IL24. PCR and southern blotting confirmed IL24 integration into the rDNA loci in four of 68 iPSC clones. We then differentiated a high expressing IL24-iPSC clone into MSCs (IL24-iMSCs) that showed higher expression of IL24 in culture supernatants and in cell lysates than control iMSCs. IL24-iMSCs efficiently differentiated into osteoblasts, chondrocytes and adipocytes. Functionally, IL24-iMSCs induced in vitro apoptosis in B16-F10 melanoma cells more efficiently than control iMSCs when co-cultured in Transwell assays. In vivo tumor xenograft studies in mice demonstrated that IL24-iMSCs inhibited melanoma growth more than control iMSCs did. Immunofluorescence and histochemical analysis showed larger necrotic areas and cell nuclear aggregation in tumors with IL24-iMSCs than control iMSCs, indicating that IL24-iMSCs inhibited tumor growth by inducing apoptosis. These findings demonstrate efficient transformation of iPSCs through gene targeting with non-viral vectors into a rDNA locus. The ability of these genetically modified MSCs to inhibit in vivo melanoma growth is suggestive of the clinical potential of autologous cell therapy in cancer.

Mesenchymal stem cells (MSCs) are a promising cellular vehicle for transferring anti-cancer factors to malignant tumors. Currently, a variety of anti-cancer agents, including the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), have been loaded into MSCs derived from a range of sources through different engineering methods. These engineered MSCs exhibit enormous therapeutic potential for various cancers. To avoid the intrinsic defects of MSCs derived from tissues and the potential risk of viral vectors, TRAIL was site-specifically integrated into the ribosomal DNA (rDNA) locus of human-induced pluripotent stem cells (iPSCs) using a non-viral rDNA-targeting vector and transcription activator-like effector nickases (TALENickases). These genetically modified human iPSCs were differentiated into an unlimited number of homogeneous induced MSCs (TRAIL-iMSCs) that overexpressed TRAIL in both culture supernatants and cell lysates while maintaining MSC-like characteristics over continuous passages. We found that TRAIL-iMSCs significantly induced apoptosis in A375, A549, HepG2, and MCF-7 cells in vitro. After intravenous infusion, TRAIL-iMSCs had a prominent tissue tropism for A549 or MCF-7 xenografts and significantly inhibited tumor growth through the activation of apoptotic signaling pathways without obvious side effects in tumor-bearing mice models. Altogether, our results showed that TRAIL-iMSCs have strong anti-tumor effects in vitro and in vivo on a range of cancers. This study allows for the development of an unlimited number of therapeutic gene-targeted MSCs with stable quality and high homogeneity for cancer therapy, thus highlighting a universal and safe strategy for stem cell-based gene therapy with high potential for clinical applications.

Alzheimer's disease (AD) is the most prevalent type of dementia and its pathology is characterized by deposition of extracellular β-amyloid plaques, intracellular neurofibrillary tangles, and extensive neuron loss. While only a few familial AD cases are due to mutations in three causative genes (APP, PSEN1, and PSEN2), the ultimate cause behind the rest of the cases, called sporadic AD, remains unknown. Current animal and cellular models of human AD, which are based on the Aβ and tau hypotheses only, partially resemble the familial AD. As a result, there is a pressing need for the development of new models providing insights into the pathological mechanisms of AD and for the discovery of ways to treat or delay the onset of the disease. Recent preclinical research suggests that stem cells can be used to model AD. Indeed, human induced pluripotent stem cells can be differentiated into disease-relevant cell types that recapitulate the unique genome of a sporadic AD patient or family member. In this review, we will first summarize the current research findings on the genetic and pathological mechanisms of AD. We will then highlight the existing induced pluripotent stem cell models of AD and, lastly, discuss the potential clinical applications in this field.

Objective To assess the diagnostic performance of a novel circulating single molecule amplification and re‐sequencing technology (cSMART) method for noninvasive prenatal testing (NIPT) of Phenylketonuria (PKU). Design Blinded NIPT analysis of pregnancies at high risk for PKU. Setting Shanghai Xinhua Hospital and Hunan Jiahui Genetics Hospital, China. Population Couples (n = 33) with a child diagnosed with PKU. Methods Trio testing for pathogenic PAH mutations was performed by Sanger sequencing. In second pregnancies, invasive prenatal diagnosis (IPD) was used to determine fetal genotypes. NIPT was performed using a PAH gene‐specific cSMART assay. Based on the plasma DNA mutation ratio relative to the fetal DNA fraction, fetal genotypes were assigned using a maximum‐likelihood algorithm. Main outcome measures Concordance of fetal genotyping results between IPD and NIPT, and the sensitivity and specificity of the NIPT assay. Results Compared with gold standard IPD results, 32 of 33 fetuses (96.97%) were accurately genotyped by NIPT. The sensitivity and specificity of the NIPT assay was 100.00% (95% CI 59.04–100.00%) and 96.15% (95% CI 80.36–99.90%), respectively. Conclusions The novel cSMART assay demonstrated high accuracy for correctly calling fetal genotypes. We propose that this test has useful clinical utility for the rapid screening of high‐risk and low‐risk pregnancies with a known history of PKU on one or both sides of the family. Tweetable abstract NIPT of couples at high risk for PKU using a full‐coverage cSMART PAH gene test.