Evaluation of the OsTIR1 and AtAFB2 AID systems for chromatin protein degradation in mammalian cells

ABSTRACTAuxin-inducible degron (AID) system is a promising tool for dynamic protein degradation. In mammalian cells, this approach has become indispensable to study fundamental molecular functions, such as replication, chromatin dynamics or transcription, that are otherwise difficult to dissect. We present evaluation of the two prominent AID systems based on OsTIR1 and AtAFB2 auxin receptor F-box proteins (AFBs). We analyzed degradation dynamics of cohesin/condensin complexes subunits in mouse embryonic stem cells (mRad21, mSMC2, mCapH, mCapH2) and human haploid HAP1 line (hRad21, hSMC2). Double antibiotic selection helped to achieve high homozygous AID targeting efficiency for all genes, ranging from 11 to 77%. We found that the main challenge for successful protein degradation is obtaining cell clones with high and stable AFB expression levels due to mosaic expression of AFBs, which also tends to decline with passages in the absence of constant puromycin selection, even at the AAVS1 safe-harbor locus. Comparing two AFBs, we found that OsTIR1 system showed weak dynamics of protein degradation. At the same time, AtAFB2 approach was very efficient even in random integration. Other factors such as degradation dynamics and low basal depletion were also in favor of AtAFB2 system. Our main conclusion is that repeated addition of puromycin to culture medium prevents AtAFB2 silencing and restores auxin sensitivity, facilitating robust protein degradation. We hope that our report will be useful for researchers that plan to establish AID method in their lab.

Gene Editing of the Human Pklr Gene in Human Hematopoietic Progenitors to Correct Pyruvate Kinase Deficiency

Pyruvate Kinase Deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene. This gene encodes the erythroid specific Pyruvate Kinase (RPK) enzyme, implying that this defective enzyme fails to produce normal levels of ATP and consequently, erythrocytes from PKD patients show an energetic imbalance. Hematopoietic stem cell gene therapy using gene editing will be the safest approach to treat PKD patients. However, its direct application in hematopoietic progenitor cells (HPCs) is challenging. Different gene editing approaches are being explored to correct PKD, either by Knock-In of an optimized cDNA sequence in the first introns of the gene or by site-specific correction using ssODN. While Knock-In strategy will allow the treatment of most PKD mutations, an ssODN-mediated gene editing will correct specific PKD patient-mutations. In our Knock-In system, a recombination matrix carrying an exon 3-11 partial codon optimized version of RPK cDNA and a puromycin selection cassette is inserted in the second intron of the PKLR gene assisted by TALEN® nucleases. We have previously shown that this approach was effective to correct PKD phenotype in PKDiPSC lines. Here, we have tested the feasibility of our approach in hematopoietic progenitors. Thus, the therapeutic matrix together with specific TALEN® for the second intron of PKLR was electroporated in purified cord blood CD34+ from healthy donors. Cells were then expanded and puromycin selected to enrich the population for gene edited ones. Up to 96% of the colony forming units that survived the puromycin selection showed the specific integration of the donor cassette. After transplantation into immunodeficient NSG mice, a low though detectable percentage of gene edited cells was . Additionally, CRISPR-Cas9 site-specific correction is being developed by combining guide RNAs directed against PKD patient's mutation and precise ssODN to restore RPK protein. Overall, our data show that gene editing in engraftable HPCs is feasible, although further improvements are needed to increase efficiency of this gene therapy appraoch prior to consider its therapeutic application in PKD patients. Disclosures Gouble: Cellectis: Employment. Galetto:Cellectis SA: Employment. Poirot:Cellectis: Employment.

C/Ebpβ Inhibits Apoptosis in Hematopoietic Cells Via Interaction with NF-κb p50.

The CCAAT/enhancer binding protein (C/EBP) family of proteins bind DNA via their C-terminal basic region – leucine zipper (BR-LZ or bZIP) domains and activate transcription via N-terminal trans-activation domains. We previously demonstrated that C/EBPα inhibits apoptosis when the immature hematopoietic Ba/F3 cell line is withdrawn from IL-3 and that a C/EBPα BR mutant that does not bind NF-κB p50 but retains the ability to bind NF-κB p65 is inactive. We have now sought to extend these findings to C/EBPβ. We first created two point mutations in C/EBPβ, R240G and ΔK253. The C/EBPβ(R240G) is analogous to the C/EBPα(R300G) BR variant that does not bind NF-κB p50 or inhibit apoptosis, and C/EBPβ(ΔK253) is analogous to the C/EBPα(ΔK313) LZ variant that does not bind DNA but retains the ability to interact with NF-κB p50 and inhibit apoptosis. In co-ip experiments using transiently transfected 293T extracts, both C/EBPβ and C/EBPβ(ΔK253) interacted with NF-κB p50, whereas C/EBPβ(R240G) did not, as predicted. In contrast, all three isoforms bound NF-κB p65. And in gel shift assay, C/EBPβ bound a consensus C/EBP site, whereas neither C/EBPβ(R240G) nor C/EBPβ(ΔK253) bound DNA, again as expected. Ba/F3 cell lines were then generated stably expressing C/EBPβ-ER, C/EBP(R240G)-ER, or C/EBPβ(ΔK253)-ER proteins, via transduction with pBabePuro retroviral vectors and puromycin selection of pooled populations. Western blot analysis confirmed similar expression of each transgene. Ba/F3 lines were removed from IL-3 and cultured with or without estradiol, to activate the ER fusion proteins. Remarkably, both CEBPβ-ER and C/EBPβ(ΔK253)-ER prolonged survival of the cells during the first 48 hrs after IL-3 withdrawal, similar to the activity of C/EBPα-ER, whereas C/EBPβ(R240G)-ER was ineffective. Current efforts focus on identifying genetic targets of C/EBPβ:NF-κB p50 cooperation relevant to the observed inhibition of apoptosis, on assessing activities of C/EBPβ and it variants in normal hematopoietic cells isolated from adult marrow, and on determining the activities of the long and short (LAP and LIP) C/EBPβ translational isoforms. Overall, the finding that C/EBPβ inhibits apoptosis in cooperation with NF-κB p50 is significant due to the expression of both C/EBPβ and activated NF-κB in several human lymphoma subsets and in solid tumors such as breast cancer. Targeting this interaction with small molecules may provide a novel therapeutic approach for these malignancies.

Endothelial cells genetically selected from differentiating mouse embryonic stem cells incorporate at sites of neovascularization in vivo

Large scale purification of endothelial cells is of great interest as it could improve tissue transplantation, reperfusion of ischemic tissues and treatment of pathologies in which an endothelial cell dysfunction exists. In this study, we describe a novel genetic approach that selects for endothelial cells from differentiating embryonic stem (ES) cells. Our strategy is based on the establishment of ES-cell clones that carry an integrated puromycin resistance gene under the control of a vascular endothelium-specific promoter, tie-1. Using EGFP as a reporter gene, we first confirmed the endothelial specificity of the tie-1 promoter in the embryoid body model and in cells differentiated in 2D cultures. Subsequently, tie-1-EGFP ES cells were used as recipients for the tie-1-driven puror transgene. The resulting stable clones were expanded and differentiated for seven days in the presence of VEGF before puromycin selection. As expected, puromycin-resistant cells were positive for EGFP and also expressed several endothelial markers, including CD31, CD34,VEGFR-1, VEGFR-2, Tie-1, VE-cadherin and ICAM-2. Release from the puromycin selection resulted in the appearance of α-smooth muscle actin-positive cells. Such cells became more numerous when the population was cultured on laminin-1 or in the presence of TGF-β1, two known inducers of smooth muscle cell differentiation. The hypothesis that endothelial cells or their progenitors may differentiate towards a smooth muscle cell phenotype was further supported by the presence of cells expressing both CD31 andα-smooth muscle actin markers. Finally, we show that purified endothelial cells can incorporate into the neovasculature of transplanted tumors in nude mice. Taken together, these results suggest that application of endothelial lineage selection to differentiating ES cells may become a useful approach for future pro-angiogenic and endothelial cell replacement therapies.