Targeted biallelic integration of an inducible Caspase 9 suicide gene for safer iPSC-based cellular therapies

The teratoma forming potential of pluripotent stem cells (PSCs), and genetic aberrations that may lead to tumor formation from PSC derivatives, are considered as a major safety risk for cellular therapies. Introduction of inducible suicide genes as synthetic fail-safe systems has been proposed to minimize these risks. Recent research challenged the usefulness of such systems even for targeted introduction via accurate gene editing approaches. Apparently transgene silencing and elimination of a HTK suicide gene through 'loss-of-heterozygosity' (LoH) led to cell clones that escaped the induced suicide. We have introduced an inducible Caspase9 (iCasp-9) suicide gene into induced pluripotent stem cells (iPSCs), that has already been applied clinically in other settings. The iCASP9 gene coupled to a red nuclear GFP variant under control of the CAG promoter was inserted into the AAVS1 locus, either monoallelic or homozygous on both alleles. Efficient induction of apoptosis in vitro could be induced via treatment of iCASP9 iPSCs with two chemical inducers of dimerization (CID) at different concentrations for 24 hours. While NODSCID mice after transplantation of undifferentiated monoallelic iCASP9 iPSCs under the kidney capsule developed teratomas, CID treatment for three days led to rapid shrinking of such tumor structures. In individual mice, however, that received monoallelic iCASP9 iPSCs, tumor-like human tissue could be detected after CID treatment. Further in vitro experiments confirmed that in very rare subclones monoallelic iCASP9 hiPSCs lost transgene expression and can became resistant to CID induction in vitro with frequencies of ~ 3x10^-8. Analysis of CID-resistant subclones identified either elimination of the transgene, presumably via LoH, or via methylation of the CAG promoter as underlying mechanism. In contrast, we never observed any CID resistant escapees form biallelic iCASP9 iPSC clones, even after treatment of up to 0,5x10^9 iPSCs. This observation further argues for LoH as underlying mechanism of transgene elimination in monoallelic clones and suggests that CAG promoter methylation on both alleles represent independent events. In conclusion, biallelic integration of an iCASP9 safety switch in the AAVS1 locus allows for efficient induction of cellular suicide and may substantially increase the safety level of iPSC-based therapies. We propose that safety levels should be calculated by relating the observed frequencies of clonal escapees to clinically relevant cell numbers, i.e. cell number in tumors of a size that is readily detectable by modern imaging approaches.

Toward Development of Neuron Specific Transduction After Systemic Delivery of Viral Vectors

Widespread transduction of the CNS with a single, non-invasive systemic injection of adeno-associated virus is now possible due to the creation of blood-brain barrier-permeable capsids. However, as these capsids are mutants of AAV9, they do not have specific neuronal tropism. Therefore, it is necessary to use genetic tools to restrict expression of the transgene to neuronal tissues. Here we compare the strength and specificity of two neuron-specific promoters, human synapsin 1 and mouse calmodulin/calcium dependent kinase II, to the ubiquitous CAG promoter. Administration of a high titer of virus is necessary for widespread CNS transduction. We observed the neuron-specific promoters drive comparable overall expression in the brain to the CAG promoter. Furthermore, the neuron-specific promoters confer significantly less transgene expression in peripheral tissues compared with the CAG promoter. Future experiments will utilize these delivery platforms to over-express the Alzheimer-associated pathological proteins amyloid-beta and tau to create mouse models without transgenesis.

Select Porcine Elongation Factor 1α Sequences Mediate Stable High-Level and Upregulated Expression of Heterologous Genes in Porcine Cells in Response to Primate Serum

Genetically engineered (GE) pigs with various combinations of genetic profiles have been developed using heterologous promoters. This study aimed to identify autologous promoters for high and ubiquitous expression of xenotransplantation relevant genes in GE pigs. A 1.4 kb upstream regulatory sequence of porcine elongation factor 1α (pEF1α) gene was selected and isolated for use as a promoter. Activity of the pEF1α promoter was subsequently compared with that of the cytomegalovirus (CMV) promoter, CMV enhancer/chicken β-actin (CAG) promoter, and human EF1α (hEF1α) promoter in different types of pig-derived cells. Comparative analysis of luciferase and mutant human leukocyte antigen class E-F2A-β-2 microglobulin (HLA-E) expression driven by pEF1α, CMV, CAG, and hEF1α promoters revealed the pEF1α promoter mediated comparable expression levels with those of the CAG promoter in porcine ear skin fibroblasts (PEFs) and porcine kidney-15 (PK-15) cells, but lower than those of the CAG promoter in porcine aortic endothelial cells (PAECs). The pEF1α promoter provided long-term stable HLA-E expression in PEFs, but the CAG promoter failed to sustain those levels of expression. For xenogeneic serum-induced cytotoxicity assays, the cells were cultured for several hours in growth medium supplemented with primate serum. Notably, the pEF1α promoter induced significant increases in luciferase and HLA-E expression in response to primate serum in PAECs compared with those driven by the CAG promoter, suggesting the pEF1α promoter could regulate temporal expression of heterologous genes under xenogeneic-cytotoxic conditions. These results suggest the pEF1α promoter may be valuable for development of GE pigs spatiotemporally and stably expressing immunomodulatory genes for xenotransplantation.

CaMKIIα Expressing Neurons to Report Activity-Related Endogenous Hypoxia upon Motor-Cognitive Challenge

We previously introduced the brain erythropoietin (EPO) circle as a model to explain the adaptive ‘brain hardware upgrade’ and enhanced performance. In this fundamental circle, brain cells, challenged by motor-cognitive tasks, experience functional hypoxia, triggering the expression of EPO among other genes. We attested hypoxic cells by a transgenic reporter approach under the ubiquitous CAG promoter, with Hif-1α oxygen-dependent degradation-domain (ODD) fused to CreERT2-recombinase. To specifically focus on the functional hypoxia of excitatory pyramidal neurons, here, we generated CaMKIIα-CreERT2-ODD::R26R-tdTomato mice. Behavioral challenges, light-sheet microscopy, immunohistochemistry, single-cell mRNA-seq, and neuronal cultures under normoxia or hypoxia served to portray these mice. Upon complex running wheel performance as the motor-cognitive task, a distinct increase in functional hypoxic neurons was assessed immunohistochemically and confirmed three-dimensionally. In contrast, fear conditioning as hippocampal stimulus was likely too short-lived to provoke neuronal hypoxia. Transcriptome data of hippocampus under normoxia versus inspiratory hypoxia revealed increases in CA1 CaMKIIα-neurons with an immature signature, characterized by the expression of Dcx, Tbr1, CaMKIIα, Tle4, and Zbtb20, and consistent with accelerated differentiation. The hypoxia reporter response was reproduced in vitro upon neuronal maturation. To conclude, task-associated activity triggers neuronal functional hypoxia as a local and brain-wide reaction mediating adaptive neuroplasticity. Hypoxia-induced genes such as EPO drive neuronal differentiation, brain maturation, and improved performance.

AAV1 is the optimal viral vector for optogenetic experiments in pigeons (Columba livia)

Although optogenetics has revolutionized rodent neuroscience, it is still rarely used in other model organisms as the efficiencies of viral gene transfer differ between species and comprehensive viral transduction studies are rare. However, for comparative research, birds offer valuable model organisms as they have excellent visual and cognitive capabilities. Therefore, the following study establishes optogenetics in pigeons on histological, physiological, and behavioral levels. We show that AAV1 is the most efficient viral vector in various brain regions and leads to extensive anterograde and retrograde ChR2 expression when combined with the CAG promoter. Furthermore, transient optical stimulation of ChR2 expressing cells in the entopallium decreases pigeons’ contrast sensitivity during a grayscale discrimination task. This finding demonstrates causal evidence for the involvement of the entopallium in contrast perception as well as a proof of principle for optogenetics in pigeons and provides the groundwork for various other methods that rely on viral gene transfer in birds.

Growth Retardation of Poorly Transfectable Tumor by Multiple Injections of Plasmids Encoding PE40 Based Targeted Toxin Complexed with Polyethylenimine

Background:: One of the approaches to cancer gene therapy relies on tumor transfection with DNA encoding toxins under the control of tumor-specific promoters. Methods:: Here, we used DNA plasmids encoding very potent anti-ERBB2 targeted toxin, driven by the human telomerase promoter or by the ubiquitous CAG promoter (pTERT-ETA and pCAG-ETA) and linear polyethylenimine to target cancer cells. Results:: We showed that the selectivity of cancer cell killing by the pTERT-ETA plasmid is highly dependent upon the method of preparation of DNA-polyethylenimine complexes. After adjustment of complex preparation protocol, cell lines with high activity of telomerase promoter can be selectively killed by transfection with the pTERT-ETA plasmid. We also showed that cells transfected with pTERT-ETA and pCAG-ETA plasmids do not exert any detectable bystander effect in vitro. Conclusion:: Despite this, three intratumoral injections of a plasmid-polyethylenimine complex resulted in substantial growth retardation of a poorly transfectable D2F2/E2 tumor in mice. There were no significant differences in anti-tumor properties between DNA constructs with telomerase or CAG promoters in vivo.

Reprogramming progressive cells display low CAG promoter activity

There is wide variability in the propensity of somatic cells to reprogram into pluripotency in response to the Yamanaka factors. How to segregate these variability to enrich for cells of specific traits that reprogram efficiently remains challenging. Here we report that the variability in reprogramming propensity is associated with the activity of the MKL1/SRF transcription factor and concurs with small cell size as well as rapid cell cycle. Reprogramming progressive cells can be prospectively identified by their low activity of a widely used synthetic promoter, CAG. CAGlow cells arise and expand during cell cycle acceleration in the early reprogramming culture of both mouse and human fibroblasts. Our work illustrate a molecular scenario underlying the distinct reprogramming propensities and demonstrate a convenient practical approach for their enrichment.