Over the last two decades, several attempts to generate packaging cells for lentiviral vectors (LV) have been made. Despite different technologies, no packaging clone is currently employed in clinical trials. We developed a new strategy for LV stable production based on the HEK-293T progenitor cells; the sequential insertion of the viral genes by integrating vectors; the constitutive expression of the viral components; and the RD114-TR envelope pseudotyping. We generated the intermediate clone PK-7 expressing constitutively gag/pol and rev genes and, by adding tat and rd114-tr genes, the stable packaging cell line RD2-MolPack, which can produce LV carrying any transfer vector (TV). Finally, we obtained the RD2-MolPack-Chim3 producer clone by transducing RD2-MolPack cells with the TV expressing the anti-HIV transgene Chim3. Remarkably, RD114-TR pseudovirions have much higher potency when produced by stable compared with transient technology. Most importantly, comparable transduction efficiency in hematopoietic stem cells (HSC) is obtained with 2-logs less physical particles respect to VSV-G pseudovirions produced by transient transfection. Altogether, RD2-MolPack technology should be considered a valid option for large-scale production of LV to be used in gene therapy protocols employing HSC, resulting in the possibility of downsizing the manufacturing scale by about 10-fold in respect to transient technology.
Background:The human Val66Met polymorphism in brain-derived neurotrophic factor (BDNF), a key factor in neuroplasticity, synaptic function, and cognition, has been implicated in the pathophysiology of neuropsychiatric and neurodegenerative disorders. BDNF is encoded by multiple transcripts with distinct regulation and localization, but the impact of the Val66Met polymorphism on BDNF regulation remains unclear.Methods:In BDNF Val66Met knock-in mice, which recapitulate the phenotypic hallmarks of individuals carrying the BDNFMet allele, we measured expression levels, epigenetic changes at promoters, and dendritic trafficking of distinct BDNF transcripts using quantitative PCR, chromatin immunoprecipitation (ChIP), and in situ hybridization.Results:BDNF-4 and BDNF-6 transcripts were reduced in BDNFMet/Met mice, compared with BDNFVal/Val mice. ChIP for acetyl-histone H3, a marker of active gene transcription, and trimethyl-histone-H3-Lys27 (H3K27me3), a marker of gene repression, showed higher H3K27me3 binding to exon 5, 6, and 8 promoters in BDNFMet/Met. The H3K27 methyltransferase enhancer of zeste homolog 2 (EZH2) is involved in epigenetic regulation of BDNF expression, because in neuroblastoma cells BDNF expression was increased both by short interference RNA for EZH2 and incubation with 3-deazaneplanocin A, an inhibitor of EZH2. In situ hybridization for BDNF-2, BDNF-4, and BDNF-6 after pilocarpine treatment showed that BDNF-6 transcript was virtually absent from distal dendrites of the CA1 and CA3 regions in BDNFMet/Met mice, while no changes were found for BDNF-2 and BDNF-4.Conclusions:Impaired BDNF expression and dendritic targeting in BDNFMet/Met mice may contribute to reduced regulated secretion of BDNF at synapses, and may be a specific correlate of pathology in individuals carrying the Met allele.
To date, gene therapy with transiently derived lentivectors has been very successful to cure rare infant genetic diseases. However, transient manufacturing is unfeasible to treat adult malignancies because large vector lots are required. By contrast, stable manufacturing is the best option for high-incidence diseases since it reduces the production cost, which is the major current limitation to scale up the transient methods. We have previously developed the proprietary RD2-MolPack technology for the stable production of second-generation lentivectors, based on the RD114-TR envelope. Of note, opposite to vesicular stomatitis virus glycoprotein (VSV-G) envelope, RD114-TR does not need inducible expression thanks to lack of toxicity. Here, we present the construction of RD2- and RD3-MolPack cells for the production of self-inactivating lentivectors expressing green fluorescent protein (GFP) as a proof-of-concept of the feasibility and safety of this technology before its later therapeutic exploitation. We report that human T lymphocytes transduced with self-inactivating lentivectors derived from RD3-MolPack cells or with self-inactivating VSV-G pseudotyped lentivectors derived from transient transfection show identical T-cell memory differentiation phenotype and comparable transduction efficiency in all T-cell subsets. RD-MolPack technology represents, therefore, a straightforward tool to simplify and standardize lentivector manufacturing to engineer T-cells for frontline immunotherapy applications.
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 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. This study addresses the need to improve the manufacturing process and the antitumor activity of CD44v6-specific CAR T cells by defining the optimal structure of a spacer region derived from the extracellular domain of the human low affinity nerve growth factor receptor (LNGFR). We tailored the LNGFR spacer to modulate CAR length in order to efficiently recognize distal or proximal epitopes and to allow selection of transduced CAR T cells by the use of clinical grade validated manufacturing systems. The different LNGFR spacers investigated in this study are responsible for the generation of CAR T cells with a different memory phenotype, which is mainly related to the level of CAR expression and the extent of the associated tonic signaling. In particular, the CD44v6-NWN2.CAR T cells are enriched in central memory cells and show improved in vitro functions in term of killing capability, and in vivo antitumor activity against hematological and solid tumors.
Ex vivo transduction of human CD34+ hematopoietic stem/progenitor cells (hCD34+ HSPCs) and T lymphocytes is a key process that requires high efficiency and low toxicity to achieve effective clinical results. So far, several enhancers have been used to improve this process. Among them, Retronectin highly meliorates VSV-G and RD114-TR pseudotyped lentiviral vector delivery in hCD34+ HSPCs and T lymphocytes. However, Retronectin is expensive and requires pre-coating of culture dishes or bags before cell seeding, resulting in a cumbersome procedure. Recently, an alternative transduction adjuvant has been developed, named Vectofusin-1, whose effect has been demonstrated on gene delivery to cell lines and primary hCD34+ HSPCs by lentiviral vectors pseudotyped with different envelope glycoproteins. In this study, we have focused our analysis on the effect of Vectofusin-1 on the transduction of hCD34+ HSPCs and T lymphocytes by using mostly RD114-TR pseudotyped lentivectors and clinical transduction protocols. Here, we have proved that Vectofusin-1 reproducibly enhances gene delivery to hCD34+ HSPCs and activated T cells without cell toxicity and with efficacy comparable to that of Retronectin. The use of Vectofusin-1 will therefore help to shorten and simplify clinical cell manipulation, especially if automated systems are planned for transducing large-scale clinical lots.
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 most commonly used in transiently produced LVs is the vesicular stomatitis virus glycoprotein (VSV-G) envelope, whose continuous expression is, however, toxic for stable LV producer cells. In contrast, the feline endogenous retroviral RD114-TR envelope is suitable for stable LV manufacturing, being well tolerated by producer cells under constitutive expression. We have previously reported successful, transient and stable production of LVs pseudotyped with RD114-TR for good transduction of T lymphocytes and CD34+ cells. To further improve RD114-TR-pseudotyped LV cell entry by increasing envelope expression, we codon-optimized the RD114-TR open reading frame (ORF). Here we show that, despite the RD114-TRco precursor being produced at a higher level than the wild-type counterpart, it is unexpectedly not duly glycosylated, exported to the cytosol, and processed. Correct cleavage of the precursor in the functional surface and transmembrane subunits is prevented in vivo, and, consequently, the unprocessed precursor is incorporated into LVs, making them inactive.
Brain-derived neurotrophic factor (BDNF), a neurotrophin highly expressed in the hippocampus, plays crucial roles in cognition, neuroplasticity, synaptic function, and dendritic remodeling. The common human Val66Met polymorphism of BDNF has been implicated in the pathophysiology of neuropsychiatric and neurodegenerative disorders, and in the outcome of pro-adaptive and therapeutic treatments. Altered gene-expression profile has been previously shown in BDNF Val66Met knock-in mice, which recapitulate the phenotypic hallmarks of individuals carrying the BDNF Met allele. The aim of this study was to investigate the impact of the BDNF Val66Met polymorphism in the knock-in mouse model on two hippocampal epigenetic marks for transcriptional repression and activation, respectively: trimethylation of lysine 27 on histone H3 (H3K27me3) and acetylation of histone H3 (AcH3), using a genome-wide approach. Chromatin immunoprecipitation followed by deep sequencing of immunoprecipitated DNA (ChIP-Seq) was carried out with specific antibodies for H3K27me3 and AcH3. Our results revealed broad alteration of H3K27me3 and AcH3 marks association profiles in BDNF , compared to BDNF mice. Bioinformatics analysis showed changes in several biological functions and related pathways, affected by the presence of the polymorphism. In particular, a number of networks of functional interaction contained BDNF as central node. Quantitative PCR analysis confirmed epigenetically related significant changes in the expression of five genes: Dvl1, Nos3, Reln, Lypd6, and Sh3gl2. The first three are involved in dendrite and spine remodeling, morphological features altered in BDNF mice. This work in homozygous knock-in mice shows that the human BDNF Val66Met polymorphism induces an array of histone H3 epigenetic modifications, in turn altering the expression of select genes crucial for structural and functional neuronal features.
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