Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing.

CRISPR-Cas RNA-guided endonucleases hold great promise for disrupting or correcting genomic sequences through site-specific DNA cleavage and repair. However, the lack of methods for cell- and tissue-selective delivery currently limits both research and clinical uses of these enzymes. We report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9-ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Uptake of such fluorescently labeled proteins in liver-derived cell lines HEPG2 (ASGPr+) and SKHEP (control; diminished ASGPr) was studied by live cell imaging and demonstrates increased accumulation of Cas9-ASGPrL RNP in HEPG2 cells as a result of effective ASGPr-mediated endocytosis. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor-facilitated and cell-type specific gene editing that did not rely on electroporation or the use of transfection reagents. Overall, these in vitro results validate the receptor-mediated delivery of genome-editing enzymes as an approach for cell-selective gene editing and provide a framework for future potential applications to hepatoselective gene editing in vivo.

Engineering CRISPR-Cas9 RNA-Protein Complexes for Improved Function and Delivery.

The use of CRISPR-derived, RNA-guided nucleases for genome editing has shown great promise for addressing genetic disease. Encouraging work in cell culture and animal models has demonstrated the capability of genome-editing enzymes to correct disease-causing loci, but clinical translation can only proceed once the corrective enzymes have been rendered safe, effective, and capable of being delivered to the appropriate cells. To address these needs, there has been rapid and extensive progress in the engineering of the RNA and protein components of Cas9, the most widely-used genome editor. Here we review advances in engineering of the enzyme Cas9 by altering its chemical or molecular composition. Such efforts have enhanced enzyme stability, improved capacity for delivery, augmented specificity, and broadened the horizons of genome manipulation.

Myocardial commitment from human pluripotent stem cells: Rapid production of human heart grafts.

Genome editing on human pluripotent stem cells (hPSCs) together with the development of protocols for organ decellularization opens the door to the generation of autologous bioartificial hearts. Here we sought to generate for the first time a fluorescent reporter human embryonic stem cell (hESC) line by means of Transcription activator-like effector nucleases (TALENs) to efficiently produce cardiomyocyte-like cells (CLCs) from hPSCs and repopulate decellularized human heart ventricles for heart engineering. In our hands, targeting myosin heavy chain locus (MYH6) with mCherry fluorescent reporter by TALEN technology in hESCs did not alter major pluripotent-related features, and allowed for the definition of a robust protocol for CLCs production also from human induced pluripotent stem cells (hiPSCs) in 14 days. hPSCs-derived CLCs (hPSCs-CLCs) were next used to recellularize acellular cardiac scaffolds. Electrophysiological responses encountered when hPSCs-CLCs were cultured on ventricular decellularized extracellular matrix (vdECM) correlated with significant increases in the levels of expression of different ion channels determinant for calcium homeostasis and heart contractile function. Overall, the approach described here allows for the rapid generation of human cardiac grafts from hPSCs, in a total of 24 days, providing a suitable platform for cardiac engineering and disease modeling in the human setting.

Accumulation of instability in serial differentiation and reprogramming of parthenogenetic human cells.

Human leukocyte antigen-homozygous parthenogenetic stem cells (pSC) could provide a source of progenitors for regenerative medicine, lowering the need for immune suppression in patients. However, the high level of homozygosis and the lack of a paternal genome might pose a safety challenge for their therapeutic use, and no study so far has evaluated the spread and significance of gene expression changes across serial potency changes in these cells. We performed serial rounds of differentiation and reprogramming to assess pSC gene expression stability, likely of epigenetic source. We first derived pSC from activated MII oocytes, and differentiated them to parthenogenetic mesenchymal stem cells (pMSC). We then proceeded to induce pluripotency in pMSC by over expression of the four transcription factors Oct4, Sox2, Klf4 and c-Myc. pMSC-derived iPS (piPS) were further differentiated into secondary pMSC (pMSC-II). At every potency change, we characterized the obtained lines both molecularly and by functional differentiation, and performed an extensive genome-wide expression study by microarray analysis. Although overall gene expression of parthenogenetic cells resembled that of potency-matched biparental lines, significantly broader changes were brought about upon secondary differentiation of piPS to pMSC-II compared with matched biparental controls; our results highlight the effect of the interplay of epigenetic reprogramming on a monoparental background, as well as the importance of heterozygosis and biparental imprinting for stable epigenetic reprogramming.

Generation of feeder-free pig induced pluripotent stem cells without Pou5f1.

The pig represents an ideal large-animal model, intermediate between rodents and humans, for the preclinical assessment of emerging cell therapies. As no validated pig embryonic stem (pES) cell lines have been derived so far, pig induced pluripotent stem cells (piPSCs) should offer an alternative source of undifferentiated cells to advance regenerative medicine research from bench to clinical trial. We report here for the first time the derivation of piPSCs from adult fibroblast with only three transcription factors: Sox2 (sex determining region Y-box 2), Klf4 (Krüppel-like factor 4), and c-Myc (avian myelocytomatosis viral oncogene homolog). We have been able to demonstrate that exogenous Pou5f1 (POU domain class 5 transcription factor 1; abbreviated as Octamer-4: Oct4) is dispensable to achieve and maintain pluripotency in the generation of piPSCs. To the best of our knowledge, this is also the first report of somatic reprogramming in any species without the overexpression, either directly or indirectly, of Oct4. Moreover, we were able to generate piPSCs without the use of feeder cells, approaching thus xeno-free conditions. Our work paves the way for the derivation of clinical grade piPSCs for regenerative medicine.