Immune tolerance promotion by LSEC-specific lentiviral vector-mediated expression of the transgene regulated by the stabilin-2 promoter.

Liver sinusoidal endothelial cells (LSECs) are specialized endocytic cells that clear the body from blood-borne pathogens and waste macromolecules through scavenger receptors (SRs). Among the various SRs expressed by LSECs is stabilin-2 (STAB2), a class H SR that binds to several ligands, among which endogenous coagulation products. Given the well-established tolerogenic function of LSECs, we asked whether the STAB2 promoter (STAB2p) would enable us to achieve LSEC-specific lentiviral vector (LV)-mediated transgene expression, and whether the expression of this transgene would be maintained over the long term due to tolerance induction. Here, we show that STAB2p ensures LSEC-specific green fluorescent protein (GFP) expression by LV in the absence of a specific cytotoxic CD8+ T cell immune response, even in the presence of GFP-specific CD8+ T cells, confirming the robust tolerogenic function of LSECs. Finally, we show that our delivery system can partially and permanently restore FVIII activity in a mouse model of severe hemophilia A without the formation of anti-FVIII antibodies. Overall, our findings establish the suitability of STAB2p for long-term LSEC-restricted expression of therapeutic proteins, such as FVIII, or to achieve antigen-specific immune tolerance in auto-immune diseases.

Deciphering the Ets-1/2-mediated transcriptional regulation of F8 gene identifies a minimal F8 promoter for hemophilia A gene therapy.

A major challenge in the development of a gene therapy for hemophilia A (HA) is the selection of cell type- or tissue-specific promoters to ensure factor VIII (FVIII) expression without eliciting an immune response. As liver sinusoidal endothelial cells (LSECs) are the major FVIII source, understanding the transcriptional F8 regulation in these cells would help optimize the minimal F8 promoter (pF8) to efficiently drive FVIII expression. In silico analyses predicted several binding sites (BS) for the E26 transformation-specific (Ets) transcription factors Ets-1 and Ets-2 in the pF8. Reporter assays demonstrated a significant up-regulation of pF8 activity by Ets-1 or Ets-1/Est-2 combination, while Ets2 alone was ineffective. Moreover, Ets-1/Ets-2-DNA binding domain mutants (DBD) abolished promoter activation only when the Ets-1 DBD was removed, suggesting that pF8 up-regulation may occur through Ets-1/Ets-2 interaction with Ets-1 bound to DNA. pF8 carrying Ets-BS deletions unveiled two Ets-BS essential for pF8 activity and response to Ets overexpression. Lentivirus-mediated delivery of GFP or FVIII cassettes driven by the shortened promoters led to GFP expression mainly in endothelial cells in the liver and to long-term FVIII activity without inhibitor formation in HA mice. These data strongly support the potential application of these promoters in HA gene therapy.

Identification and functional characterization of a novel splicing variant in the F8 coagulation gene causing severe hemophilia A.

BACKGROUND: We have identified a synonymous F8 variation in a severe hemophilia A (HA) patient who developed inhibitors following factor VIII (FVIII) prophylaxis. The unreported c.6273 G > A variant targets the consensus splicing site of exon 21. OBJECTIVES: To determine the impact of c.6273 G > A nucleotide substitution on F8 splicing and its translated protein. METHODS: Patient peripheral blood mononuclear cells were isolated and differentiated into monocyte-derived macrophages (MDMs). FVIII distribution in cell compartments was evaluated by immunofluorescence. The splicing of mutated exon 21 was assessed by exon trapping. Identified FVIII splicing variants were generated by site-directed mutagenesis, inserted into a lentiviral vector (LV) to transduce Chinese hamster ovary (CHO) cells, and inject into B6/129 HA-mice. FVIII activity was assessed by activated partial thromboplastin time, whereas anti-FVIII antibodies and FVIII antigen, by ELISA. RESULTS: HA-MDMs demonstrated a predominant retention of FVIII around the endoplasmic reticulum. Exon trapping revealed the production of two isoforms: one retaining part of intron 21 and the other skipping exon 21. These variants, predicted to truncate FVIII in the C1 domain, were detected in the patient. CHO cells transduced with the two FVIII transcripts confirmed protein retention and absence of the C2 domain. HA mice injected with LV carrying FVIII mutants, partially recovered FVIII activity without the appearance of anti-FVIII antibodies. CONCLUSIONS: Herein, we demonstrate the aberrant impact of a FVIII synonymous mutation on its transcription, activity, and pathological outcomes. Our data underline the importance of increasing the knowledge regarding the functional consequences of F8 mutations and their link to inhibitor development and an effective replacement therapy.

FVIII expression by its native promoter sustains long-term correction avoiding immune response in hemophilic mice.

Here we describe a successful gene therapy approach for hemophilia A (HA), using the natural F8 promoter (pF8) to direct gene replacement to factor VIII (FVIII)-secreting cells. The promoter sequence and the regulatory elements involved in the modulation of F8 expression are still poorly characterized and biased by the historical assumption that FVIII expression is mainly in hepatocytes. Bioinformatic analyses have highlighted an underestimated complexity in gene expression at this locus, suggesting an activation of pF8 in more cell types than those previously expected. C57Bl/6 mice injected with a lentiviral vector expressing green fluorescent protein (GFP) under the pF8 (lentiviral vector [LV].pF8.GFP) confirm the predominant GFP expression in liver sinusoidal endothelial cells, with a few positive cells detectable also in hematopoietic organs. Therapeutic gene delivery (LV.pF8.FVIII) in hemophilic C57/Bl6 and 129-Bl6 mice successfully corrected the bleeding phenotype, rescuing up to 25% FVIII activity, using a codon-optimized FVIII, with sustained activity for the duration of the experiment (1 year) without inhibitor formation. Of note, LV.pF8.FVIII delivery in FVIII-immunized HA mice resulted in the complete reversion of the inhibitor titer with the recovery of therapeutic FVIII activity. Depletion of regulatory T cells (Tregs) in LV-treated mice allowed the formation of anti-FVIII antibodies, indicating a role for Tregs in immune tolerance induction. The significant blood loss reduction observed in all LV.pF8.FVIII-treated mice 1 year after injection confirmed the achievement of a long-term phenotypic correction. Altogether, our results highlight the potency of pF8-driven transgene expression to correct the bleeding phenotype in HA, as well as potentially in other diseases in which an endothelial-specific expression is required.

Clinical impact of small TP53 mutated subclones in chronic lymphocytic leukemia.

TP53 mutations are strong predictors of poor survival and refractoriness in chronic lymphocytic leukemia (CLL) and have direct implications for disease management. Clinical information on TP53 mutations is limited to lesions represented in >20% leukemic cells. Here, we tested the clinical impact and prediction of chemorefractoriness of very small TP53 mutated subclones. The TP53 gene underwent ultra-deep-next generation sequencing (NGS) in 309 newly diagnosed CLL. A robust bioinformatic algorithm was established for the highly sensitive detection of few TP53 mutated cells (down to 3 out of ∼1000 wild-type cells). Minor subclones were validated by independent approaches. Ultra-deep-NGS identified small TP53 mutated subclones in 28/309 (9%) untreated CLL that, due to their very low abundance (median allele frequency: 2.1%), were missed by Sanger sequencing. Patients harboring small TP53 mutated subclones showed the same clinical phenotype and poor survival (hazard ratio = 2.01; P = .0250) as those of patients carrying clonal TP53 lesions. By longitudinal analysis, small TP53 mutated subclones identified before treatment became the predominant population at the time of CLL relapse and anticipated the development of chemorefractoriness. This study provides a proof-of-principle that very minor leukemia subclones detected at diagnosis are an important driver of the subsequent disease course.

Alterations of negative regulators of cytokine signalling in immunodeficiency-related non-Hodgkin lymphoma.

We investigated immunodeficiency-related non-Hodgkin lymphoma for the presence of molecular alterations affecting negative regulators of the Janus family protein tyrosine kinase/signal transducer and activator of transcription pathway. Protein tyrosine phosphatase, non-receptor type 6/Src homology 2-containing tyrosine phosphatase-1 epigenetic silencing was recurrent in primary effusion lymphoma (100%), and diffuse large B-cell lymphoma (63%), with a higher prevalence in the non-germinal centre subtype, and was associated with the activation of the Janus family protein tyrosine kinase/signal transducer and activator of transcription 3 pathway. Suppressor of cytokine signalling (SOCS)1 and SOCS3 epigenetic silencing were occasionally detected, whereas SOCS1 was frequently mutated in diffuse large B-cell lymphoma and polymorphic post-transplant lymphoproliferative disorders, possibly as a cause of aberrant somatic hypermutation. However, the mutation profile of the coding region of the gene was different from that expected from the aberrant somatic hypermutation process, suggesting that, at least in some cases, SOCS1 mutations may have been selected for their functional activity.

The coding genome of splenic marginal zone lymphoma: activation of NOTCH2 and other pathways regulating marginal zone development.

Splenic marginal zone lymphoma (SMZL) is a B cell malignancy of unknown pathogenesis, and thus an orphan of targeted therapies. By integrating whole-exome sequencing and copy-number analysis, we show that the SMZL exome carries at least 30 nonsilent gene alterations. Mutations in NOTCH2, a gene required for marginal-zone (MZ) B cell development, represent the most frequent lesion in SMZL, accounting for ∼20% of cases. All NOTCH2 mutations are predicted to cause impaired degradation of the NOTCH2 protein by eliminating the C-terminal PEST domain, which is required for proteasomal recruitment. Among indolent B cell lymphoproliferative disorders, NOTCH2 mutations are restricted to SMZL, thus representing a potential diagnostic marker for this lymphoma type. In addition to NOTCH2, other modulators or members of the NOTCH pathway are recurrently targeted by genetic lesions in SMZL; these include NOTCH1, SPEN, and DTX1. We also noted mutations in other signaling pathways normally involved in MZ B cell development, suggesting that deregulation of MZ B cell development pathways plays a role in the pathogenesis of ∼60% SMZL. These findings have direct implications for the treatment of SMZL patients, given the availability of drugs that can target NOTCH, NF-κB, and other pathways deregulated in this disease.

Disruption of BIRC3 associates with fludarabine chemorefractoriness in TP53 wild-type chronic lymphocytic leukemia.

The genetic lesions identified to date do not fully recapitulate the molecular pathogenesis of chronic lymphocytic leukemia (CLL) and do not entirely explain the development of severe complications such as chemorefractoriness. In the present study, BIRC3, a negative regulator of noncanonical NF-κB signaling, was investigated in different CLL clinical phases. BIRC3 lesions were absent in monoclonal B-cell lymphocytosis (0 of 63) and were rare in CLL at diagnosis (13 of 306, 4%). Conversely, BIRC3 disruption selectively affected 12 of 49 (24%) fludarabine-refractory CLL cases by inactivating mutations and/or gene deletions that distributed in a mutually exclusive fashion with TP53 abnormalities. In contrast to fludarabine-refractory CLL, progressive but fludarabine-sensitive patients were consistently devoid of BIRC3 abnormalities, suggesting that BIRC3 genetic lesions associate specifically with a chemorefractory phenotype. By actuarial analysis in newly diagnosed CLL (n = 306), BIRC3 disruption identified patients with a poor outcome similar to that associated with TP53 abnormalities and exerted a prognostic role that was independent of widely accepted clinical and genetic risk factors. Consistent with the role of BIRC3 as a negative regulator of NF-κB, biochemical studies revealed the presence of constitutive noncanonical NF-κB activation in fludarabine-refractory CLL patients harboring molecular lesions of BIRC3. These data identify BIRC3 disruption as a recurrent genetic lesion of high-risk CLL devoid of TP53 abnormalities.

Mutations of NOTCH1 are an independent predictor of survival in chronic lymphocytic leukemia.

Analysis of the chronic lymphocytic leukemia (CLL) coding genome has recently disclosed that the NOTCH1 proto-oncogene is recurrently mutated at CLL presentation. Here, we assessed the prognostic role of NOTCH1 mutations in CLL. Two series of newly diagnosed CLL were used as training (n = 309) and validation (n = 230) cohorts. NOTCH1 mutations occurred in 11.0% and 11.3% CLL of the training and validation series, respectively. In the training series, NOTCH1 mutations led to a 3.77-fold increase in the hazard of death and to shorter overall survival (OS; P < .001). Multivariate analysis selected NOTCH1 mutations as an independent predictor of OS after controlling for confounding clinical and biologic variables. The independent prognostic value of NOTCH1 mutations was externally confirmed in the validation series. The poor prognosis conferred by NOTCH1 mutations was attributable, at least in part, to shorter treatment-free survival and higher risk of Richter transformation. Although NOTCH1 mutated patients were devoid of TP53 disruption in more than 90% cases in both training and validation series, the OS predicted by NOTCH1 mutations was similar to that of TP53 mutated/deleted CLL. NOTCH1 mutations are an independent predictor of CLL OS, tend to be mutually exclusive with TP53 abnormalities, and identify cases with a dismal prognosis.