O-GlcNAcylation enhances CPS1 catalytic efficiency for ammonia and promotes ureagenesis

Life-threatening hyperammonemia occurs in both inherited and acquired liver diseases affecting ureagenesis, the main pathway for detoxification of neurotoxic ammonia in mammals. Protein O-GlcNAcylation is a reversible and nutrient-sensitive post-translational modification using as substrate UDP-GlcNAc, the end-product of hexosamine biosynthesis pathway. Here we show that increased liver UDP-GlcNAc during hyperammonemia increases protein O-GlcNAcylation and enhances ureagenesis. Mechanistically, O-GlcNAcylation on specific threonine residues increased the catalytic efficiency for ammonia of carbamoyl phosphate synthetase 1 (CPS1), the rate-limiting enzyme in ureagenesis. Pharmacological inhibition of O-GlcNAcase, the enzyme removing O-GlcNAc from proteins, resulted in clinically relevant reductions of systemic ammonia in both genetic (hypomorphic mouse model of propionic acidemia) and acquired (thioacetamide-induced acute liver failure) mouse models of liver diseases. In conclusion, by fine-tuned control of ammonia entry into ureagenesis, hepatic O-GlcNAcylation of CPS1 increases ammonia detoxification and is a novel target for therapy of hyperammonemia in both genetic and acquired diseases

Patient derived CAR T cell expressing the Suicide gene iC9 represent a safe approach in case of CAR+ leukemia relapse

Use of chimeric antigen receptor T cells (CAR T) for treatment of relapsing/refractory acute lymphoblastic leukemia (ALL) has been accompanied by exciting clinical results. However, lentiviral vector-based clinical approaches revealed a potential risk of relapse associated with CD19-/CAR+ leukemic clones. To verify if also use of a γ-retroviral platform may lead to emergence of CD19-/CAR+ leukemia cells, either peripheral blood or bone marrow-derived mononuclear cells of patients with >40% of blasts at diagnosis (CD45dim+/CD34+/CD19+/CD22+/CD10+), were transduced with a second-generation CAR.CD19 in frame with a suicide gene (namely, inducible caspase 9, iC9). Patient-derived CAR-T cells showed a phenotype not significantly different from that seen in healthy-donors. In particular, both flow-cytometry analysis and Real-Time-quantitative PCR failed to identify leukemic cells in the final CAR-T cell product generated from B-cells precursor ALL (Bcp-ALL) patients. In vitro studies also showed that CD19+ leukemic cell lines transduced with the iC9.CAR.CD19 vector, mimicking the model in which leukemic cells could be genetically modified with CAR, were efficiently eliminated by activation of iC9. Moreover, by applying a long-term time-frame in vivo mouse experiment, and we provided evidences that use of the γ-retroviral CAR platform is not associated with the expansion of leukemia cells characterized by CAR positivity. Taken together these data suggest that the use of a γ-retroviral platform with the addition of the iC9 suicide gene allows to obtain a highly functional and safe CAR.T product, even when the production starts from biological material characterized by heavy contamination with leukemia blasts

Liver-directed gene therapy for ornithine aminotransferase deficiency

: Gyrate atrophy of choroid and retina (GACR) is a chorioretinal degeneration caused by pathogenic variants in the gene encoding ornithine aminotransferase (OAT), an enzyme mainly expressed in liver. Affected patients have increased ornithine concentrations in blood and other body fluids and develop progressive constriction of vision fields leading to blindness. Current therapies are unsatisfactory and better treatments are highly needed. In two mouse models of OAT deficiency that recapitulates biochemical and retinal changes of GACR, we investigated the efficacy of an intravenously injected serotype 8 adeno-associated (AAV8) vector expressing OAT under the control of a hepatocyte-specific promoter. Following injections, OAT-deficient mice showed reductions of ornithine concentrations in blood and eye cups compared with control mice injected with a vector expressing green fluorescent protein. AAV-injected mice showed improved electroretinogram response and partial restoration of retinal structure up to one-year post-injection. In summary, hepatic OAT expression by AAV8 vector was effective at correction of hyperornithinemia and improved function and structure of the retina. In conclusion, this study provides proof-of-concept of efficacy of liver-directed AAV-mediated gene therapy of GACR

Choice of costimulatory domains and of cytokines determines CAR T-cell activity in neuroblastoma

Chimeric antigen receptor (CAR) T-cell therapy has been shown to be dramatically effective in the treatment of B-cell malignancies. However, there are still substantial obstacles to overcome, before similar responses can be achieved in patients with solid tumors. We evaluated both in vitro and in a preclinical murine model the efficacy of different 2nd and 3rd generation CAR constructs targeting GD2, a disial-ganglioside expressed on the surface of neuroblastoma (NB) tumor cells. In order to address potential safety concerns regarding clinical application, an inducible safety switch, namely inducible Caspase-9 (iC9), was also included in the vector constructs. Our data indicate that a 3rd generation CAR incorporating CD28.4-1BB costimulatory domains is associated with improved anti-tumor efficacy as compared with a CAR incorporating the combination of CD28.OX40 domains. We demonstrate that the choice of 4-1BB signaling results into significant amelioration of several CAR T-cell characteristics, including: 1) T-cell exhaustion, 2) basal T-cell activation, 3) in vivo tumor control and 4) T-cell persistence. The fine-tuning of T-cell culture conditions obtained using IL7 and IL15 was found to be synergic with the CAR.GD2 design in increasing the anti-tumor activity of CAR T cells. We also demonstrate that activation of the suicide gene iC9, included in our construct without significantly impairing neither CAR expression nor anti-tumor activity, leads to a prompt induction of apoptosis of GD2.CAR T cells. Altogether, these findings are instrumental in optimizing the function of CAR T-cell products to be employed in the treatment of children with NB

Adoptive immunotherapy using prame-specific t cells in medulloblastoma

Medulloblastoma is the most frequent malignant childhood brain tumor with a high morbidity. Identification of new therapeutic targets would be instrumental in improving patient outcomes. We evaluated the expression of the tumor-associated antigen PRAME in biopsies from 60 patients with medulloblastoma. PRAME expression was detectable in 82% of tissues independent of molecular and histopathologic subgroups. High PRAME expression also correlated with worse overall survival. We next investigated the relevance of PRAME as a target for immunotherapy. Medulloblastoma cells were targeted using genetically modified T cells with a PRAME-specific TCR (SLL TCR T cells). SLL TCR T cells efficiently killed medulloblastoma HLA-A-02+ DAOY cells as well as primary HLA-A-02+ medulloblastoma cells. Moreover, SLL TCR T cells controlled tumor growth in an orthotopic mouse model of medulloblastoma. To prevent unexpected T-cell- related toxicity, an inducible caspase-9 (iC9) gene was introduced in frame with the SLL TCR; this safety switch triggered prompt elimination of genetically modified T cells. Altogether, these data indicate that T cells genetically modified with a high-affinity, PRAME-specific TCR and iC9 may represent a promising innovative approach for treating patients with HLA-A-02+ medulloblastoma. Significance: These findings identify PRAME as a medulloblastoma tumor-associated antigen that can be targeted using genetically modified T cells

Efficacy of third-party chimeric antigen receptor modified peripheral blood natural killer cells for adoptive cell therapy of B cell precursor acute lymphoblastic leukemia

We developed an innovative and efficient, feeder-free culture method to genetically modify and expand peripheral blood-derived NK cells with high proliferative capacity, while preserving the responsiveness of their native activating receptors. Activated peripheral blood NK cells were efficiently transduced by a retroviral vector, carrying a second-generation CAR targeting CD19. CAR expression was demonstrated across the different NK-cell subsets. CAR.CD19-NK cells display higher antileukemic activity toward CD19+ cell lines and primary blasts obtained from patients with B-cell precursor ALL compared with unmodified NK cells. In vivo animal model data showed that the antileukemia activity of CAR.CD19-NK cell is superimposable to that of CAR-T cells, with a lower xenograft toxicity profile. These data support the feasibility of generating feeder-free expanded, genetically modified peripheral blood NK cells for effective “off-the-shelf” immuno-gene-therapy, while their innate alloreactivity can be safely harnessed to potentiate allogeneic cell therapy

O-GlcNAcylation enhances CPS1 catalytic efficiency for ammonia and promotes ureagenesis

Life-threatening hyperammonemia occurs in both inherited and acquired liver diseases affecting ureagenesis, the main pathway for detoxification of neurotoxic ammonia in mammals. Protein O-GlcNAcylation is a reversible and nutrient-sensitive post-translational modification using as substrate UDP-GlcNAc, the end-product of hexosamine biosynthesis pathway. Here we show that increased liver UDP-GlcNAc during hyperammonemia increases protein O-GlcNAcylation and enhances ureagenesis. Mechanistically, O-GlcNAcylation on specific threonine residues increased the catalytic efficiency for ammonia of carbamoyl phosphate synthetase 1 (CPS1), the rate-limiting enzyme in ureagenesis. Pharmacological inhibition of O-GlcNAcase, the enzyme removing O-GlcNAc from proteins, resulted in clinically relevant reductions of systemic ammonia in both genetic (hypomorphic mouse model of propionic acidemia) and acquired (thioacetamide-induced acute liver failure) mouse models of liver diseases. In conclusion, by fine-tuned control of ammonia entry into ureagenesis, hepatic O-GlcNAcylation of CPS1 increases ammonia detoxification and is a novel target for therapy of hyperammonemia in both genetic and acquired diseases

Metastatic group 3 medulloblastoma is driven by PRUNE1 targeting NME1-TGF-β-OTX2-SNAIL via PTEN inhibition

Genetic modifications during development of paediatric groups 3 and 4 medulloblastoma are responsible for their highly metastatic properties and poor patient survival rates. PRUNE1 is highly expressed in metastatic medulloblastoma group 3, which is characterized by TGF-β signalling activation, c-MYC amplification, and OTX2 expression. We describe the process of activation of the PRUNE1 signalling pathway that includes its binding to NME1, TGF-β activation, OTX2 upregulation, SNAIL (SNAI1) upregulation, and PTEN inhibition. The newly identified small molecule pyrimido-pyrimidine derivative AA7.1 enhances PRUNE1 degradation, inhibits this activation network, and augments PTEN expression. Both AA7.1 and a competitive permeable peptide that impairs PRUNE1/NME1 complex formation, impair tumour growth and metastatic dissemination in orthotopic xenograft models with a metastatic medulloblastoma group 3 cell line (D425-Med cells). Using whole exome sequencing technology in metastatic medulloblastoma primary tumour cells, we also define 23 common 'non-synonymous homozygous' deleterious gene variants as part of the protein molecular network of relevance for metastatic processes. This PRUNE1/TGF-β/OTX2/PTEN axis, together with the medulloblastoma-driver mutations, is of relevance for future rational and targeted therapies for metastatic medulloblastoma group 3.Genetic modifications during development of paediatric groups 3 and 4 medulloblastoma are responsible for their highly metastatic properties and poor patient survival rates. PRUNE1 is highly expressed in metastatic medulloblastoma group 3, which is characterized by TGF-β signalling activation, c-MYC amplification, and OTX2 expression. We describe the process of activation of the PRUNE1 signalling pathway that includes its binding to NME1, TGF-β activation, OTX2 upregulation, SNAIL (SNAI1) upregulation, and PTEN inhibition. The newly identified small molecule pyrimido-pyrimidine derivative AA7.1 enhances PRUNE1 degradation, inhibits this activation network, and augments PTEN expression. Both AA7.1 and a competitive permeable peptide that impairs PRUNE1/NME1 complex formation, impair tumour growth and metastatic dissemination in orthotopic xenograft models with a metastatic medulloblastoma group 3 cell line (D425-Med cells). Using whole exome sequencing technology in metastatic medulloblastoma primary tumour cells, we also define 23 common 'non-synonymous homozygous' deleterious gene variants as part of the protein molecular network of relevance for metastatic processes. This PRUNE1/TGF-β/OTX2/PTEN axis, together with the medulloblastoma-driver mutations, is of relevance for future rational and targeted therapies for metastatic medulloblastoma group 3. 10.1093/brain/awy039-video1 awy039media1 574205353400