[How safe is gene therapy? : Second death after Duchenne therapy]. / Wie sicher ist die Gentherapie? : Zweiter Todesfall nach Therapie der Duchenne-Muskeldystrophie.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a severe monogenic hereditary disease with early manifestation and a progressive course. Treatment options have so far been limited. Gene therapy opens up new options for DMD patients. OBJECTIVES: Against the background of a further death following DMD gene therapy, the side effects and risks of the gene therapeutics already approved or undergoing clinical trials will be evaluated and alternative gene therapeutics will be described. Based thereon, the future of DMD gene therapy will be discussed. CURRENT DATA: For the first time, in June 2023, delandistrogene moxeparvovec (SRP-9001), a gene replacement therapy based on an adeno-associated virus (AAV) vector, was approved in the USA for children aged 4-5 years with DMD. Other promising gene therapies are in preclinical development or clinical trials, including CRISPR/Cas9-mediated strategies to restore dystrophin expression. Two deaths following DMD gene therapy with high-dose AAV vectors were attributed to AAV-mediated immune responses. The pre-existing disease underlying the therapy is most likely involved in the fatal AAV toxicity. CONCLUSIONS: Although gene therapy applications of AAV vectors are generally considered safe, the systemic administration of high vector doses can lead to severe side effects with a potentially fatal outcome in individual patients, especially after activation of the immune system. In the future, new methods for immunosuppression, reduction of AAV dose and alternative vectors will therefore increasingly come to the fore.

CD19-targeted chimeric antigen receptor T cell therapy in two patients with multiple sclerosis.

BACKGROUND: Progressive multiple sclerosis (MS) is characterized by compartmentalized smoldering neuroinflammation caused by the proliferation of immune cells residing in the central nervous system (CNS), including B cells. Although inflammatory activity can be prevented by immunomodulatory therapies during early disease, such therapies typically fail to halt disease progression. CD19 chimeric antigen receptor (CAR)-T cell therapies have revolutionized the field of hematologic malignancies. Although generally considered efficacious, serious adverse events associated with CAR-T cell therapies such as immune effector cell-associated neurotoxicity syndrome (ICANS) have been observed. Successful use of CD19 CAR-T cells in rheumatic diseases like systemic lupus erythematosus and neuroimmunological diseases like myasthenia gravis have recently been observed, suggesting possible application in other autoimmune diseases. METHODS: Here, we report the first individual treatment with a fully human CD19 CAR-T cell therapy (KYV-101) in two patients with progressive MS. FINDINGS: CD19 CAR-T cell administration resulted in acceptable safety profiles for both patients. No ICANS was observed despite detection of CD19 CAR-T cells in the cerebrospinal fluid. In case 1, intrathecal antibody production in the cerebrospinal fluid decreased notably after CAR-T cell infusion and was sustained through day 64. CONCLUSIONS: CD19 CAR-T cell administration in progressive MS resulted in an acceptable safety profile. CAR-T cell presence and expansion were observed in the cerebrospinal fluid without clinical signs of neurotoxicity, which, along with intrathecal antibody reduction, indicates expansion-dependent effects of CAR-T cells on CD19+ target cells in the CNS. Larger clinical studies assessing CD19 CAR-T cells in MS are warranted. FUNDING: Both individual treatments as well the generated data were not based on external funding.

CD45-Directed CAR-T Cells with CD45 Knockout Efficiently Kill Myeloid Leukemia and Lymphoma Cells In Vitro Even after Extended Culture.

BACKGROUND: CAR-T cell therapy has shown impressive results and is now part of standard-of-care treatment of B-lineage malignancies, whereas the treatment of myeloid diseases has been limited by the lack of suitable targets. CD45 is expressed on almost all types of blood cells including myeloid leukemia cells, but not on non-hematopoietic tissue, making it a potential target for CAR-directed therapy. Because of its high expression on T and NK cells, fratricide is expected to hinder CD45CAR-mediated therapy. Due to its important roles in effector cell activation, signal transduction and cytotoxicity, CD45 knockout aimed at preventing fratricide in T and NK cells has been expected to lead to considerable functional impairment. METHODS: CD45 knockout was established on T and NK cell lines using CRISPR/Cas9-RNPs and electroporation, and the successful protocol was transferred to primary T cells. A combined protocol was developed enabling CD45 knockout and retroviral transduction with a third-generation CAR targeting CD45 or CD19. The functionality of CD45ko effector cells, CD45ko/CD45CAR-T and CD45ko/CD19CAR-T cells was studied using proliferation as well as short- and long-term cytotoxicity assays. RESULTS: As expected, the introduction of a CD45-CAR into T cells resulted in potent fratricide that can be avoided by CD45 knockout. Unexpectedly, the latter had no negative impact on T- and NK-cell proliferation in vitro. Moreover, CD45ko/CD45CAR-T cells showed potent cytotoxicity against CD45-expressing AML and lymphoma cell lines in short-term and long-term co-culture assays. A pronounced cytotoxicity of CD45ko/CD45CAR-T cells was maintained even after four weeks of culture. In a further setup, we confirmed the conserved functionality of CD45ko cells using a CD19-CAR. Again, the proliferation and cytotoxicity of CD45ko/CD19CAR-T cells showed no differences from those of their CD45-positive counterparts in vitro. CONCLUSIONS: We report the efficient production of highly and durably active CD45ko/CAR-T cells. CD45 knockout did not impair the functionality of CAR-T cells in vitro, irrespective of the target antigen. If their activity can be confirmed in vivo, CD45ko/CD45CAR-T cells might, for example, be useful as part of conditioning regimens prior to stem cell transplantation.

HLA-DPA1*02:01~B1*01:01 is a risk haplotype for primary sclerosing cholangitis mediating activation of NKp44+ NK cells.

OBJECTIVE: Primary sclerosing cholangitis (PSC) is characterised by bile duct strictures and progressive liver disease, eventually requiring liver transplantation. Although the pathogenesis of PSC remains incompletely understood, strong associations with HLA-class II haplotypes have been described. As specific HLA-DP molecules can bind the activating NK-cell receptor NKp44, we investigated the role of HLA-DP/NKp44-interactions in PSC. DESIGN: Liver tissue, intrahepatic and peripheral blood lymphocytes of individuals with PSC and control individuals were characterised using flow cytometry, immunohistochemical and immunofluorescence analyses. HLA-DPA1 and HLA-DPB1 imputation and association analyses were performed in 3408 individuals with PSC and 34 213 controls. NK cell activation on NKp44/HLA-DP interactions was assessed in vitro using plate-bound HLA-DP molecules and HLA-DPB wildtype versus knock-out human cholangiocyte organoids. RESULTS: NKp44+NK cells were enriched in livers, and intrahepatic bile ducts of individuals with PSC showed higher expression of HLA-DP. HLA-DP haplotype analysis revealed a highly elevated PSC risk for HLA-DPA1*02:01~B1*01:01 (OR 1.99, p=6.7×10-50). Primary NKp44+NK cells exhibited significantly higher degranulation in response to plate-bound HLA-DPA1*02:01-DPB1*01:01 compared with control HLA-DP molecules, which were inhibited by anti-NKp44-blocking. Human cholangiocyte organoids expressing HLA-DPA1*02:01-DPB1*01:01 after IFN-γ-exposure demonstrated significantly increased binding to NKp44-Fc constructs compared with unstimulated controls. Importantly, HLA-DPA1*02:01-DPB1*01:01-expressing organoids increased degranulation of NKp44+NK cells compared with HLA-DPB1-KO organoids. CONCLUSION: Our studies identify a novel PSC risk haplotype HLA-DP A1*02:01~DPB1*01:01 and provide clinical and functional data implicating NKp44+NK cells that recognise HLA-DPA1*02:01-DPB1*01:01 expressed on cholangiocytes in PSC pathogenesis.

IL-10 dampens antitumor immunity and promotes liver metastasis via PD-L1 induction.

BACKGROUND & AIMS: The liver is one of the organs most commonly affected by metastasis. The presence of liver metastases has been reported to be responsible for an immunosuppressive microenvironment and diminished immunotherapy efficacy. Herein, we aimed to investigate the role of IL-10 in liver metastasis and to determine how its modulation could affect the efficacy of immunotherapy in vivo. METHODS: To induce spontaneous or forced liver metastasis in mice, murine cancer cells (MC38) or colon tumor organoids were injected into the cecum or the spleen, respectively. Mice with complete and cell type-specific deletion of IL-10 and IL-10 receptor alpha were used to identify the source and the target of IL-10 during metastasis formation. Programmed death ligand 1 (PD-L1)-deficient mice were used to test the role of this checkpoint. Flow cytometry was applied to characterize the regulation of PD-L1 by IL-10. RESULTS: We found that Il10-deficient mice and mice treated with IL-10 receptor alpha antibodies were protected against liver metastasis formation. Furthermore, by using IL-10 reporter mice, we demonstrated that Foxp3+ regulatory T cells (Tregs) were the major cellular source of IL-10 in liver metastatic sites. Accordingly, deletion of IL-10 in Tregs, but not in myeloid cells, led to reduced liver metastasis. Mechanistically, IL-10 acted on Tregs in an autocrine manner, thereby further amplifying IL-10 production. Furthermore, IL-10 acted on myeloid cells, i.e. monocytes, and induced the upregulation of the immune checkpoint protein PD-L1. Finally, the PD-L1/PD-1 axis attenuated CD8-dependent cytotoxicity against metastatic lesions. CONCLUSIONS: Treg-derived IL-10 upregulates PD-L1 expression in monocytes, which in turn reduces CD8+ T-cell infiltration and related antitumor immunity in the context of colorectal cancer-derived liver metastases. These findings provide the basis for future monitoring and targeting of IL-10 in colorectal cancer-derived liver metastases. IMPACT AND IMPLICATIONS: Liver metastasis diminishes the effectiveness of immunotherapy and increases the mortality rate in patients with colorectal cancer. We investigated the role of IL-10 in liver metastasis formation and assessed its impact on the effectiveness of immunotherapy. Our data show that IL-10 is a pro-metastatic factor involved in liver metastasis formation and that it acts as a regulator of PD-L1. This provides the basis for future monitoring and targeting of IL-10 in colorectal cancer-derived liver metastasis.

Chaperone-mediated autophagy in neuronal dendrites utilizes activity-dependent lysosomal exocytosis for protein disposal.

The complex morphology of neurons poses a challenge for proteostasis because the majority of lysosomal degradation machinery is present in the cell soma. In recent years, however, mature lysosomes were identified in dendrites, and a fraction of those appear to fuse with the plasma membrane and release their content to the extracellular space. Here, we report that dendritic lysosomes are heterogeneous in their composition and that only those containing lysosome-associated membrane protein (LAMP) 2A and 2B fuse with the membrane and exhibit activity-dependent motility. Exocytotic lysosomes dock in close proximity to GluN2B-containing N-methyl-D-aspartate-receptors (NMDAR) via an association of LAMP2B to the membrane-associated guanylate kinase family member SAP102/Dlg3. NMDAR-activation decreases lysosome motility and promotes membrane fusion. We find that chaperone-mediated autophagy is a supplier of content that is released to the extracellular space via lysosome exocytosis. This mechanism enables local disposal of aggregation-prone proteins like TDP-43 and huntingtin.

Donor Lymphocyte Infusion and Molecular Monitoring for Relapsed Myelofibrosis After Hematopoietic Cell Transplantation.

Hematopoietic cell transplantation (HCT) is a curative approach for myelofibrosis patients, but relapse is a major cause of treatment failure. We investigated the effect of donor lymphocyte infusion (DLI) in 37 patients with molecular (n = 17) or hematological relapse (n = 20) after HCT. Patients received median of 2 (range, 1-5) cumulative DLI (total of 91 infusions). Median starting dose was 1 × 106 cells/kg, escalated by half-log ≥6 weeks if no response nor graft-versus-host disease (GvHD) occurred. Median time to first DLI was 40 weeks for molecular relapse versus 145 weeks for hematological relapse. Overall molecular complete response (mCR) at any time was 73% (n = 27) and was significantly higher for initial molecular relapse (88%) versus hematological relapse (60%; P = 0.05). The 6-year overall survival was 77% versus 32% (P = 0.03). Acute GvHD 2-4 occurred in 22% and half of the patients achieved mCR without any GvHD. All patients who relapsed from mCR achieved after first DLI could be salvaged with subsequent DLI, showing long-term survival. No second HCT was needed for molecular relapse versus 6 for hematological relapse. This comprehensive and largest study to date suggests molecular monitoring together with DLI as standard of care and a crucial approach to achieve excellent outcomes in relapsed myelofibrosis.

Deep Characterization and Comparison of Different Retrovirus-like Particles Preloaded with CRISPR/Cas9 RNPs.

The CRISPR/Cas system has a broad range of possible medical applications, but its clinical translation has been hampered, particularly by the lack of safe and efficient vector systems mediating the short-term expression of its components. Recently, different virus-like particles (VLPs) have been introduced as promising vectors for the delivery of CRISPR/Cas genome editing components. Here, we characterized and directly compared three different types of retrovirus-based (R) VLPs, two derived from the γ-retrovirus murine leukemia virus (gRVLPs and "enhanced" egRVLPs) and one from the lentivirus human immunodeficiency virus, HIV (LVLPs). First, we unified and optimized the production of the different RVLPs. To ensure maximal comparability of the produced RVLPs, we adapted several assays, including nanoparticle tracking analysis (NTA), multi-parametric imaging flow cytometry (IFC), and Cas9-ELISA, to analyze their morphology, surface composition, size, and concentration. Next, we comparatively tested the three RVLPs targeting different genes in 293T model cells. Using identical gRNAs, we found egRVLPs to mediate the most efficient editing. Functional analyses indicated better cargo (i.e., Cas9) transfer and/or release as the underlying reason for their superior performance. Finally, we compared on- and off-target activities of the three RVLPs in human-induced pluripotent stem cells (hiPSC) exploiting the clinically relevant C-C motif chemokine receptor 5 (CCR5) as the target. Again, egRVLPs facilitated the highest, almost 100% knockout rates, importantly with minimal off-target activity. In conclusion, in direct comparison, egRVLPs were the most efficient RVLPs. Moreover, we established methods for in-depth characterization of VLPs, facilitating their validation and thus more predictable and safe application.

Microglia colonize the developing brain by clonal expansion of highly proliferative progenitors, following allometric scaling.

Microglia arise from the yolk sac and enter the brain during early embryogenesis. Upon entry, microglia undergo in situ proliferation and eventually colonize the entire brain by the third postnatal week in mice. However, the intricacies of their developmental expansion remain unclear. Here, we characterize the proliferative dynamics of microglia during embryonic and postnatal development using complementary fate-mapping techniques. We demonstrate that the developmental colonization of the brain is facilitated by clonal expansion of highly proliferative microglial progenitors that occupy spatial niches throughout the brain. Moreover, the spatial distribution of microglia switches from a clustered to a random pattern between embryonic and late postnatal development. Interestingly, the developmental increase in microglial numbers follows the proportional growth of the brain in an allometric manner until a mosaic distribution has been established. Overall, our findings offer insight into how the competition for space may drive microglial colonization by clonal expansion during development.

Spleen volume and length determined by computed tomography impact outcome after allogeneic stem cell transplantation for myelofibrosis.

Splenomegaly is a hallmark of myelofibrosis (MF), and reports on the impact of spleen size on the outcome of allo-HSCT have been conflicting, possibly due to differences in methods of assessment. We retrospectively analysed the impact of spleen volume and length measured by computed tomography on allo-HSCT outcome in 93 patients, 74% of whom had prior ruxolitinib treatment. Median spleen volume and length were 1.58 dm3 and 20 cm, respectively. We found a strong correlation between spleen volume and length (Pearson's r = 0.95, p < 0.001), Spearman (rho = 0.96, p < 0.001). After a median follow-up of 41.7 months, 5-year overall and disease-free survival were 66% and 59%, respectively. Spleen size did not impact overall survival or non-relapse mortality. Larger spleen volume and length as continuous variables were associated with slower platelet and leucocyte engraftment and a higher risk of disease relapse in univariate and multivariate analyses. Spleen length measured precisely by imaging is a good surrogate for spleen volume. In the era of JAK inhibitors, larger spleen size reflects advanced disease in MF and is associated with an increased risk of relapse but has no impact on non-relapse mortality and overall survival after allo-HSCT.

Molecular monitoring of T-cell kinetics and migration in severe neurotoxicity after real-world CD19-specific chimeric antigen receptor T cell therapy.

CD19-specific chimeric antigen receptor (CD19-CAR) T-cell therapies mediate durable responses in late-stage B-cell malignancies, but can be complicated by a potentially severe immune effector cell-associated neurotoxicity syndrome (ICANS). Despite broad efforts, the precise mechanisms of ICANS are not entirely known, and resistance to current ICANSdirected therapies (especially corticosteroids) has been observed. Recent data suggest that inflammatory cytokines and/or targeting of cerebral CD19-expressing pericytes can disrupt the blood-brain barrier and facilitate influx of immune cells, including CAR T cells. However, specific tools for CD19-CAR T-cell analysis within often minute samples of cerebrospinal fluid (CSF) are not broadly available. Here, we applied our recently developed digital polymerase chain reaction assays to monitor CD19-CAR T-cell kinetics in CSF and blood in real-world patients with neurotoxicity. Consistently, we observed a CAR T-cell enrichment within CSF in ICANS patients with further progressive accumulation despite intense corticosteroid- containing immuno-chemotherapies in a subset of patients with prolonged and therapy-resistant grade 3-4 neurotoxicity. We used next-generation T-cell receptor-b sequencing to assess the repertoire of treatment-refractory cells. Longitudinal analysis revealed a profound skewing of the T-cell receptor repertoire, which at least partly reflected selective expansion of infused T-cell clones. Interestingly, a major fraction of eventually dominating hyperexpanded T-cell clones were of non-CAR T-cell derivation. These findings hint to a role of therapy-refractory T-cell clones in severe ICANS development and prompt future systematic research to determine if CAR T cells may serve as 'door openers' and to further characterize both CAR-positive and non-CAR T cells to interrogate the transcriptional signature of these possibly pathologic T cells.

Generating Patient-Derived HCC Cell Lines Suitable for Predictive In Vitro and In Vivo Drug Screening by Orthotopic Transplantation.

Hepatocellular carcinoma (HCC) results in high mortality due to ineffective systemic therapy. Human immortalized cell lines are commonly used to study anti-tumor effects in the context of new anti-tumor therapies and tumor biology. As immortalized cell lines have limited biological relevance and heterogeneity compared to primary cells, patient-derived tumor tissues, and corresponding immune cells are the gold standards for studying the complexity of individual tumor entities. However, culturing primary HCC cells has a low success rate. Here, we aimed to establish a reproducible approach to preserve the patient-derived liver cancer cells for in vitro and in vivo studies. The underlying study aimed to establish an in vitro pre-screening platform to test treatment options' effectivity and dosage, e.g., for new substances, autologous modified immune cells, or combined therapies in HCC. We initially employed 15 surgical resection specimens from patients with different HCC entities for isolation and preservation. The isolated liver cancer cells from four HCC-diagnosed patients were used for orthotopic transplantation into the healthy liver of immunodeficient mice, allowing them to grow for six months before human liver cancer cells were isolated and cultured. As a result, we generated and characterized four new primary-like liver cancer cell lines. Compared to immortalized HCC cell lines, freshly generated liver cancer cells displayed individual morphologies and heterogeneous protein-level characteristics. We assessed their ability to proliferate, migrate, form spheroids, and react to common medications compared to immortalized HCC cell lines. All four liver cancer cell lines exhibit strong migration and colony-forming characteristics in vitro, comparable to extensively investigated immortalized HCC cell lines. Moreover, the four etiological different liver cancer cell lines displayed differences in the response to 5-FU, Sorafenib, Axitinib, and interferon-alpha treatment, ranking from non-responders to responders depending on the applicated medication. In sum, we generated individual patient-derived liver cancer cell lines suitable for predictive in vitro drug screenings and for xenograft transplantations to realize the in vivo investigation of drug candidates. We overcame the low cultivation success rate of liver cancer cells derived from patients and analyzed their potential to serve a pre-clinical model.

Population Pharmacokinetics of Busulfan and Its Metabolite Sulfolane in Patients with Myelofibrosis Undergoing Hematopoietic Stem Cell Transplantation.

For patients with myelofibrosis, allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the only curative treatment to date. Busulfan-based conditioning regimens are commonly used, although high inter-individual variability (IIV) in busulfan drug exposure makes individual dose selection challenging. Since data regarding the IIV in patients with myelofibrosis are sparse, this study aimed to develop a population pharmacokinetic (PopPK) model of busulfan and its metabolite sulfolane in patients with myelofibrosis. The influence of patient-specific covariates on the pharmacokinetics of drug and metabolite was assessed using non-linear mixed effects modeling in NONMEM®. We obtained 523 plasma concentrations of busulfan and its metabolite sulfolane from 37 patients with myelofibrosis. The final model showed a population clearance (CL) and volume of distribution (Vd) of 0.217 L/h/kg and 0.82 L/kg for busulfan and 0.021 L/h/kg and 0.65 L/kg for its metabolite. Total body weight (TBW) and a single-nucleotide polymorphism of glutathione-S-transferase A1 (GSTA1 SNP) displayed a significant impact on volume of distribution and metabolite clearance, respectively. This is the first PopPK-model developed to describe busulfan's pharmacokinetics in patients with myelofibrosis. Incorporating its metabolite sulfolane into the model not only allowed the characterization of the covariate relationship between GSTA1 and the clearance of the metabolite but also improved the understanding of busulfan's metabolic pathway.

High-throughput drug screening allowed identification of entry inhibitors specifically targeting different routes of SARS-CoV-2 Delta and Omicron/BA.1.

The Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2) has continuously evolved, resulting in the emergence of several variants of concern (VOCs). To study mechanisms of viral entry and potentially identify specific inhibitors, we pseudotyped lentiviral vectors with different SARS-CoV-2 VOC spike variants (D614G, Alpha, Beta, Delta, Omicron/BA.1), responsible for receptor binding and membrane fusion. These SARS-CoV-2 lentiviral pseudoviruses were applied to screen 774 FDA-approved drugs. For the assay we decided to use CaCo2 cells, since they equally allow cell entry through both the direct membrane fusion pathway mediated by TMPRSS2 and the endocytosis pathway mediated by cathepsin-L. The active molecules which showed stronger differences in their potency to inhibit certain SARS-CoV-2 VOCs included antagonists of G-protein coupled receptors, like phenothiazine-derived antipsychotic compounds such as Chlorpromazine, with highest activity against the Omicron pseudovirus. In general, our data showed that the various VOCs differ in their preferences for cell entry, and we were able to identify synergistic combinations of inhibitors. Notably, Omicron singled out by relying primarily on the endocytosis pathway while Delta preferred cell entry via membrane fusion. In conclusion, our data provide new insights into different entry preferences of SARS-CoV-2 VOCs, which might help to identify new drug targets.

Combined Targeting of AKT and mTOR Inhibits Tumor Formation of EpCAM+ and CD90+ Human Hepatocellular Carcinoma Cells in an Orthotopic Mouse Model.

The epithelial cell adhesion molecule (EpCAM) and Thy-1 cell surface antigen (CD90) have been implicated as cancer stem cell (CSC) markers in hepatocellular carcinoma (HCC). Expression of EpCAM and CD90 on HCC cells is associated with increased tumorigenicity, metastasis and poor prognosis. In this study, we demonstrate that combined treatment with AKT and mTOR inhibitors-i.e., MK2206 and RAD001-results in a synergistic reduction in proliferation of EpCAM+ and CD90+ HCC cells cultured either as adherent cells or as tumoroids in vitro. In addition, tumor growth was reduced by combined treatment with AKT and mTOR inhibitors in an orthotopic xenograft mouse model of an EpCAM+ HCC cell line (Huh7) and primary patient-derived EpCAM+ HCC cells (HCC1) as well as a CD90+ HCC-related cell line (SK-HEP1) in vivo. However, during AKT/mTOR treatment, outgrowth of therapy-resistant tumors was observed in all mice analyzed within a few weeks. Resistance was associated in most cases with restoration of AKT signaling in the tumors, intrahepatic metastases and distant metastases. In addition, an upregulation of the p38 MAPK pathway was identified in the AKT/mTOR inhibitor-resistant tumor cells by kinome profiling. The development of resistant cells during AKT/mTOR therapy was further analyzed by red-green-blue (RGB) marking of HCC cells, which revealed an outgrowth of a large number of Huh7 cells over a period of 6 months. In summary, our data demonstrate that combined treatment with AKT and mTOR inhibitors exhibits synergistic effects on proliferation of EpCAM+ as well as CD90+ HCC cells in vitro. However, the fast development of large numbers of resistant clones under AKT/mTOR therapy observed in vitro and in the orthotopic xenotransplantation mouse model in vivo strongly suggests that this therapy alone will not be sufficient to eliminate EpCAM+ or CD90+ cancer stem cells from HCC patients.