Cell-to-cell variability in inducible Caspase9-mediated cell death

AbstractiCasp9 suicide gene has been widely used as a promising killing strategy in various cell therapies. However, different cells show significant heterogeneity in response to apoptosis inducer, posing challenges in clinical applications of killing strategy. The cause of the heterogeneity remains elusive so far. Here, by simultaneously monitoring the dynamics of iCasp9 dimerization, Caspase3 activation, and cell fate in single cells, we found that the heterogeneity was mainly due to cell-to-cell variability in initial iCasp9 expression and XIAP/Caspase3 ratio. Moreover, multiple-round drugging cannot increase the killing efficiency. Instead, it will place selective pressure on protein levels, especially on the level of initial iCasp9, leading to drug resistance. We further show this resistance can be largely eliminated by combinatorial drugging with XIAP inhibitor at the end, but not at the beginning, of the multiple-round treatments. Our results unveil the source of cell fate heterogeneity and drug resistance in iCasp9-mediated cell death, which may enlighten better therapeutic strategies for optimized killing.

Increased Susceptibility of the CD57– NK Cells Expressing KIR2DL2/3 and NKG2C to iCasp9 Gene Retroviral Transduction and the Relationships with Proliferative Potential, Activation Degree, and Death Induction Response

Nowadays, the use of genetically modified NK cells is a promising strategy for cancer immunotherapy. The additional insertion of genes capable of inducing cell suicide allows for the timely elimination of the modified NK cells. Different subsets of the heterogenic NK cell population may differ in proliferative potential, in susceptibility to genetic viral transduction, and to the subsequent induction of cell death. The CD57–NKG2C+ NK cells are of special interest as potential candidates for therapeutic usage due to their high proliferative potential and certain features of adaptive NK cells. In this study, CD57– NK cell subsets differing in KIR2DL2/3 and NKG2C expression were transduced with the iCasp9 suicide gene. The highest transduction efficacy was observed in the KIR2DL2/3+NKG2C+ NK cell subset, which demonstrated an increased proliferative potential with prolonged cultivation. The increased transduction efficiency of the cell cultures was associated with the higher expression level of the HLA-DR activation marker. Among the iCasp9-transduced subsets, KIR2DL2/3+ cells had the weakest response to the apoptosis induction by the chemical inductor of dimerization (CID). Thus, KIR2DL2/3+NKG2C+ NK cells showed an increased susceptibility to the iCasp9 retroviral transduction, which was associated with higher proliferative potential and activation status. However, the complete elimination of these cells with CID is impeded.

CBMS-3 Potent bystander effect in suicide gene therapy using genetically engineered stem cells from human exfoliated deciduous teeth

HSV thymidine kinase (TK)/ganciclovir (GCV) has a long history of application in malignant glioma and we have previously demonstrated its bystander effect on gliomas using several stem cell types as a vehicle. The main reason for applying stem cells is that they have a unique tumor-trophic activity that allows them to deliver TK genes efficiently to nearby the tumor. Stem cells from human exfoliated deciduous teeth (SHED) are mesenchymal stem cells easily harvested from dental pulp and no studies have reported suicide gene therapy using SHED as a carrier for malignant gliomas. For transduction of SHED with the HSVTK gene (SHEDTK), we used HSVTK retrovirus-producing cells.In vitro experiments showed a significant migration ability of SHEDTK toward tumor-conditioned medium and representative tumor growth factors. We also detected a significant bystander effect of SHEDTK on gliomas in the presence of GCV. In vitro time-lapse imaging showed that both SHEDTK and glioma cells underwent gradual morphological apoptosis and activation of caspase 3/7 was observed in both cell types. In intracranial tumor models using nude mice, SHEDTK migrated around the U87 cell mass implanted in the contralateral hemisphere. Additionally, coculture suspensions of SHEDTK and U87-luciferase cells were xeno-transplanted followed by intraperitoneal administration of GCV for 10 days. All mice of treatment group survived for more than 100 days, whereas those treated without GCV died of tumor growth with median survival of 42 days after tumor implantation. Furthermore, pre-existing intracranial U87 model mice were injected intratumorally with SHEDTK followed by GCV administration as described above. The tumor volume was significantly reduced during the treatment period, and over-all surivial in treatment group is prolonged significantly to that of control groups. These results indicate that SHEDTK-based suicide gene therapy might offer a new promising therapeutic modality for human malignant gliomas.

Barnase-Barstar Pair: Contemporary Application in Cancer Research and Nanotechnology

Barnase is an extracellular ribonuclease secreted by Bacillus amyloliquefaciens that was originally studied as a small stable enzyme with robust folding. The identification of barnase intracellular inhibitor barstar led to the discovery of an incredibly strong protein-protein interaction. Together, barnase and barstar provide a fully genetically encoded toxin-antitoxin pair having an extremely low dissociation constant. Moreover, compared to other dimerization systems, the barnase-barstar module provides the exact one-to-one ratio of the complex components and possesses high stability of each component in a complex and high solubility in aqueous solutions without self-aggregation. The unique properties of barnase and barstar allow the application of this pair for the engineering of different variants of targeted anticancer compounds and cytotoxic supramolecular complexes. Using barnase in suicide gene therapy has also found its niche in anticancer therapy. The application of barnase and barstar in contemporary experimental cancer therapy is reflected in the review.

AVALIAÇÃO DA TERAPIA GÊNICA DO SUICÍDIO COM USO DE GENES SUICIDAS PARA O COMBATE AO CÂNCER: REVISÃO INTEGRATIVA

Introdução: Durante décadas os tratamentos quimioterápicos convencionais foram empregados no câncer devido à eficácia em matar as células tumorais. Diante disso, a terapia gênica com uso de genes suicidas surge como uma das abordagens mais inovadoras para o desenvolvimento de agentes antineoplásicos com maior seletividade tumoral. Assim, entre os genes suicidas disponíveis, a timidina quinase é investigada em vários modelos de tumor. Contudo, o uso dessa enzima apresenta limitações devido ao sistema de imunogenicidade. Recentemente, pesquisadores passaram a utilizar o sistema de gene suicida da Caspase-9 induzível, a qual apresentou resultados mais favoráveis se comparado à timidina quinase. Objetivo: Objetiva-se construir uma revisão integrativa da literatura sobre a terapia gênica do suicídio avaliando o uso de genes suicidas, sobretudo da timidina quinase e da caspase-9 induzível para o combate ao câncer. Material e Métodos: Foi realizada uma revisão integrativa de literatura por meio de uma pesquisa nos bancos de dados PubMed, Scopus e LILACS com os seguintes descritores “terapia gênica de genes suicidas”, “câncer”, “avaliação”, “timidina quinase” e “caspase-9 induzível” simultaneamente às correspondentes em inglês, em intervalo de 11 anos (2010-2021), com critérios de inclusão preestabelecidos. Para o cruzamento dos descritores, utilizou-se um protocolo com os seguintes booleanos: Gene Therapy of Suicide Gene AND Cancer AND Assessment AND Thymidine Kinase AND Inducible Caspase-9. Ao final, 4 artigos foram selecionados. Resultados: Foi selecionado 1 artigo (n = 25%) que abordava o tema da avaliação na terapia de genes suicidas. Em contrapartida, os critérios de exclusão foram estudos que não contemplavam o efeito avaliativo desses genes na terapia gênica, resumindo-se a 3 artigos selecionados (n = 75%). Com isso, observou-se que a avaliação do uso de genes suicidas ainda é uma abordagem recente, necessitando de mais estudos que contemplem essa temática. Conclusão: A avaliação da terapia gênica do suicídio pautada no uso de genes suicidas para o combate ao câncer é promissora, mesmo que recente e dos mínimos estudos publicados. Além disso, o sistema de entrega utilizando a caspase-9 induzível é mais viável do que o sistema de entrega da timidina quinase, já que esse sistema se limita à imunogenicidade do transgene viral.

CSIG-19. DISRUPTION OF DNA DAMAGE RESPONSE MODULATES THE EFFICACY OF LOCAL IMMUNOTHERAPIES IN EXPERIMENTAL GLIOMA

RATIONALE Herpes virus thymidine kinase (HSV-TK) suicide gene therapy is a well-established approach for in situ tumor cell killing after administration of ganciclovir (GCV), due to the induction of lethal DNA damage in HSV-TK expressing cells. Here we investigated the effects of HSV-TK gene delivery with a non-replicating serotype 5 adenovirus (AdTK) in murine glioblastoma models in combination with the ATR-inhibitor AZD6738 to disrupt the DNA damage response (DDR). METHODS We investigated the effects of disrupted DDR signaling on AdTK therapy in vitro using cytotoxicity, cytokine and flow cytometry assays in glioblastoma cell lines and in vivo with an orthotopic syngeneic murine glioblastoma model. Therapy response was monitored with MRI. Changes in the tumor microenvironment were analyzed with CyTOF. RESULTS The combination of AZD6738 with AdTK was synergistic in cytotoxicity assays, which was complemented by a significant increase of γH2AX foci. Complex modulations of the tumor microenvironment were observed with significantly reduced expression of PD-L1, MICA/B and the pro-tumorigenic cytokines IL1b and IL-4. In vivo, the combination with AZD6738 led to an increase in long-term surviving animals (66.7%) compared to GMCI (50%) and proved to be highly significant in contrast to untreated controls (p=0.0022). However, the combination treatment did not block the growth of tumors upon rechallenge in long-term survivors. CONCLUSION DDR signaling is crucial in the therapeutic efficacy of AdTK/GCV. It significantly enhances cytotoxicity in vitro and in vivo while having complex ramifications at the immunological level, requiring further studies to determine ideal conditions for a maximized therapeutic benefit.

Anthracycline-free tumor elimination in mice leads to functional and molecular cardiac recovery from cancer-induced alterations in contrast to long-lasting doxorubicin treatment effects

Systemic effects of advanced cancer impact on the heart leading to cardiac atrophy and functional impairment. Using a murine melanoma cancer model (B16F10 melanoma cells stably transduced with a Ganciclovir (GCV)-inducible suicide gene), the present study analysed the recovery potential of cancer-induced cardiomyopathy with or without use of doxorubicin (Dox). After Dox-free tumor elimination and recovery for 70 ± 5 days, cancer-induced morphologic, functional, metabolic and molecular changes were largely reversible in mice previously bearing tumors. Moreover, grip strength and cardiac response to angiotensin II-induced high blood pressure were comparable with healthy control mice. In turn, addition of Dox (12 mg/kg BW) to melanoma-bearing mice reduced survival in the acute phase compared to GCV-alone induced recovery, while long-term effects on cardiac morphologic and functional recovery were similar. However, Dox treatment was associated with permanent changes in the cardiac gene expression pattern, especially the circadian rhythm pathway associated with the DNA damage repair system. Thus, the heart can recover from cancer-induced damage after chemotherapy-free tumor elimination. In contrast, treatment with the cardiotoxic drug Dox induces, besides well-known adverse acute effects, long-term subclinical changes in the heart, especially of circadian clock genes. Since the circadian clock is known to impact on cardiac repair mechanisms, these changes may render the heart more sensitive to additional stress during lifetime, which, at least in part, could contribute to late cardiac toxicity.

Hypoxia-directed tumor targeting of CRISPR/Cas9 and HSV-TK suicide gene therapy using lipid nanoparticles

Hypoxia is a characteristic feature of solid tumors that contributes to tumor aggressiveness and is associated with resistance to cancer therapy. The hypoxia inducible factor-1 (HIF-1) transcription factor complex mediates hypoxia-specific gene expression by binding to hypoxia responsive element (HRE) sequences within the promoter of target genes. HRE driven expression of therapeutic cargo has been widely explored as a strategy to achieve cancer-specific gene expression. By utilizing this system, we achieve hypoxia-specific expression of two therapeutically relevant cargo elements: the Herpes Simplex Virus thymidine kinase (HSV-tk) suicide gene and the CRISPR/Cas9 nuclease. Using an expression vector containing five copies of the HRE derived from the vascular endothelial growth factor gene, we are able to show high transgene expression in cells in a hypoxic environment, similar to levels achieved using the CMV and CBh promoters. Furthermore, we are able to deliver our therapeutic cargo to tumor cells with high efficiency using plasmid packaged lipid nanoparticles (LNPs) to achieve specific killing of tumor cells in hypoxic conditions, while maintaining tight regulation with no significant changes to cell viability in normoxia.

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.