Unbiased assessment of genome integrity and purging of adverse outcomes at the target locus upon editing of CD4 T-cells for the treatment of Hyper IgM1.

Hyper IgM1 is an X-linked combined immunodeficiency caused by CD40LG mutations, potentially treatable with CD4 T-cell gene editing with Cas9 and a "one-size-fits-most" corrective template. Contrary to established gene therapies, there is limited data on the genomic alterations following long-range gene editing, and no consensus on the relevant assays. We developed drop-off digital PCR assays for unbiased detection of large on-target deletions and found them at high frequency upon editing.

Scalable GMP-compliant gene correction of CD4+ T cells with IDLV template functionally validated and .

Hyper-IgM1 is a rare X-linked combined immunodeficiency caused by mutations in the CD40 ligand () gene with a median survival of 25 years, potentially treatable with CD4+ T cell gene editing with Cas9 and a one-size-fits-most corrective donor template. Here, starting from our research-grade editing protocol, we pursued the development of a good manufacturing practice (GMP)-compliant, scalable process that allows for correction, selection and expansion of edited cells, using an integrase defective lentiviral vector as donor template. After systematic optimization of reagents and conditions we proved maintenance of stem and central memory phenotypes and expression and function of in edited healthy donor and patient cells recapitulating the physiological regulation.

Characterization and Functional Analysis of CD44v6.CAR T Cells Endowed with a New Low-Affinity Nerve Growth Factor Receptor-Based Spacer.

Effectiveness of adoptively transferred chimeric antigen receptor (CAR) T cells strongly depends on the quality of CAR-mediated interaction of the effector cells with the target antigen on tumor cells. A major role in this interaction is played by the affinity of the single-chain variable fragment (scFv) for the antigen, and by the CAR design. In particular, the spacer domain may impact on the CAR T cell function by affecting the length and flexibility of the resulting CAR.

CAR T Cells Redirected to CD44v6 Control Tumor Growth in Lung and Ovary Adenocarcinoma Bearing Mice.

The main challenge of adoptive therapy with Chimeric Antigen Receptor modified T cells (CAR T) is the application to the field of solid tumors, where the identification of a proper antigen has emerged as one of the major drawbacks to CAR T cell treatment success. CD44 is a glycoprotein involved in cell-cell and cell-matrix interactions. The isoform containing the variant domain 6 of CD44 gene (CD44v6) has been implicated in tumorigenesis, tumor cell invasion and metastasis and represents an attractive target for CAR T cell therapies.

Mechanism of Action of the Tumor Vessel Targeting Agent NGR-hTNF: Role of Both NGR Peptide and hTNF in Cell Binding and Signaling.

NGR-hTNF is a therapeutic agent for a solid tumor that specifically targets angiogenic tumor blood vessels, through the NGR motif. Its activity has been assessed in several clinical studies encompassing tumors of different histological types. The drug's activity is based on an improved permeabilization of newly formed tumor vasculature, which favors intratumor penetration of chemotherapeutic agents and leukocyte trafficking.

Codon Optimization Leads to Functional Impairment of RD114-TR Envelope Glycoprotein.

Lentiviral vectors (LVs) are a highly valuable tool for gene transfer currently exploited in basic, applied, and clinical studies. Their optimization is therefore very important for the field of vectorology and gene therapy. A key molecule for LV function is the envelope because it guides cell entry.

The tumor vessel targeting agent NGR-TNF controls the different stages of the tumorigenic process in transgenic mice by distinct mechanisms.

NGR-TNF is a vascular targeting agent in advanced clinical development, coupling tumor necrosis factor-α (TNF) with the CNGRCG peptide, which targets a CD13 isoform specifically expressed by angiogenic vessels. Antitumor efficacy of NGR-TNF has been described in different transplantation tumor models. Nevertheless, the mechanism underlying its activity is not fully understood.

Molecular dynamics reveal that isoDGR-containing cyclopeptides are true αvβ3 antagonists unable to promote integrin allostery and activation.

Ain't got that swing(-out): The cyclopeptide isoDGR is emerging as a new αvβ3 integrin binding motif. Agreement between the results of computational and biochemical studies reveals that isoDGR-containing cyclopeptides are true αvβ3 integrin antagonists that block αvβ3 in its inactive conformation (see scheme). isoDGR-based ligands may give αvβ3 antagonists without paradoxical effects.

Biochemical and functional characterization of the interaction between liprin-α1 and GIT1: implications for the regulation of cell motility.

We have previously identified the scaffold protein liprin-α1 as an important regulator of integrin-mediated cell motility and tumor cell invasion. Liprin-α1 may interact with different proteins, and the functional significance of these interactions in the regulation of cell motility is poorly known. Here we have addressed the involvement of the liprin-α1 partner GIT1 in liprin-α1-mediated effects on cell spreading and migration.

Liprin-alpha1 affects the distribution of low-affinity beta1 integrins and stabilizes their permanence at the cell surface.

Integrins mediate the interaction between cells and extracellular matrix by assembling adhesive structures that need to be dynamically modulated to allow cell motility. We have recently identified liprin-alpha1 as an essential regulator of integrin dynamics required for efficient cell motility. Here we investigated the effects of liprin-alpha1 expression on beta1 integrin receptors.

Liprin-alpha1 promotes cell spreading on the extracellular matrix by affecting the distribution of activated integrins.

Integrin activation is needed to link the extracellular matrix with the actin cytoskeleton during cell motility. Protrusion requires coordination of actin dynamics with focal-adhesion turnover. We report that the adaptor protein liprin-alpha1 is stably associated with the cell membrane.

Identification of an intramolecular interaction important for the regulation of GIT1 functions.

G-protein coupled receptor kinase-interacting protein (GIT) proteins include an N-terminal Arf GTPase-activating protein domain, and a C terminus that binds proteins regulating adhesion and motility. Given their ability to form large molecular assemblies, the GIT1 protein must be tightly regulated. However, the mechanisms regulating GIT1 functions are poorly characterized.