Interactions in vivo between the Vif protein of HIV-1 and the precursor (Pr55GAG) of the virion nucleocapsid proteins

The abnormality of viral core structure seen in vif-defective HIV-1 grown in PBMCs has suggested a role for Vif in viral morphogenesis. Using an in vivo mammalian two-hybrid assay, the interaction between Vif and the precursor (Pr55GAG) of the virion nucleocapsid proteins has been analysed. This revealed the amino-terminal (aa 1–22) and central (aa 70–100) regions of Vif to be essential for its interaction with Pr55GAG, but deletion of the carboxy-terminal (aa 158–192) region of the protein had only a minor effect on its interaction. Initial deletion studies carried out on Pr55GAG showed that a 35-amino-acid region of the protein bridging the MA(p17)–CA(p24) junction was essential for its ability to interact with Vif. Site-directed mutagenesis of a conserved tryptophan (Trp21) near the amino terminus of Vif showed it to be important for the interaction with Pr55GAG. By contrast, mutagenesis of the highly conserved YLAL residues forming part of the BC-box motif, shown to be important in Vif promoting degradation of APOBEC3G/3F, had little or no effect on the Vif–Pr55GAG interaction

Phase 1 clinical trial of CRISPR-engineered CAR19 universal T cells for treatment of children with refractory B cell leukemia

Genome editing of allogeneic T cells can provide “off-the-shelf” alternatives to autologous chimeric antigen receptor (CAR) T cell therapies. Disruption of T cell receptor α chain (TRAC) to prevent graft-versus-host disease (GVHD) and removal of CD52 (cluster of differentiation 52) for a survival advantage in the presence of alemtuzumab have previously been investigated using transcription activator–like effector nuclease (TALEN)-mediated knockout. Here, we deployed next-generation CRISPR-Cas9 editing and linked CAR expression to multiplexed DNA editing of TRAC and CD52 through incorporation of self-duplicating CRISPR guide RNA expression cassettes within the 3’ long terminal repeat of a CAR19 lentiviral vector. Three cell banks of TT52CAR19 T cells were generated and cryopreserved. A phase 1, open-label, non-randomized clinical trial was conducted and treated six children with relapsed/refractory CD19-positive B cell acute lymphoblastic leukemia (B-ALL) (NCT04557436). Lymphodepletion included fludarabine, cyclophosphamide, and alemtuzumab and was followed by a single infusion of 0.8 × 10^{6} to 2.0 × 10^{6} CAR19 T cells per kilogram with no immediate toxicities. Four of six patients infused with TT52CAR19 T cells exhibited cell expansion, achieved flow cytometric remission, and then proceeded to receive allogeneic stem cell transplantation. Two patients required biological intervention for grade II cytokine release syndrome, one patient developed transient grade IV neurotoxicity, and one patient developed skin GVHD, which resolved after transplant conditioning. Other complications were within expectations, and primary safety objectives were met. This study provides a demonstration of the feasibility, safety, and therapeutic potential of CRISPR-engineered immunotherapy

Integrin‐Targeted, Short Interfering RNA Nanocomplexes for Neuroblastoma Tumor‐Specific Delivery Achieve MYCN Silencing with Improved Survival

The authors aim to develop siRNA therapeutics for cancer that can be administered systemically to target tumors and retard their growth. The efficacy of systemic delivery of siRNA to tumors with nanoparticles based on lipids or polymers is often compromised by their rapid clearance from the circulation by the liver. Here, multifunctional cationic and anionic siRNA nanoparticle formulations are described, termed receptor‐targeted nanocomplexes (RTNs), that comprise peptides for siRNA packaging into nanoparticles and receptor‐mediated cell uptake, together with lipids that confer nanoparticles with stealth properties to enhance stability in the circulation, and fusogenic properties to enhance endosomal release within the cell. Intravenous administration of RTNs in mice leads to predominant accumulation in xenograft tumors, with very little detected in the liver, lung, or spleen. Although non‐targeted RTNs also enter the tumor, cell uptake appears to be RGD peptide‐dependent indicating integrin‐mediated uptake. RTNs with siRNA against MYCN (a member of the Myc family of transcription factors) in mice with MYCN‐amplified neuroblastoma tumors show significant retardation of xenograft tumor growth and enhanced survival. This study shows that RTN formulations can achieve specific tumor‐targeting, with minimal clearance by the liver and so enable delivery of tumor‐targeted siRNA therapeutics

In Vivo and In Vitro Functional Studies on the HIV-1 Vif Protein

Human Immunodeficiency virus type-l (HIV-l), has a number of regulatory genes in addition to the gag, pol, and env that are common to all replication competent retrovimses. It expresses six auxilialY proteins, tat, rev, Vi/, vpu, vpx and nef. Vif (Viral Infectivity Factor) is a 23 kDa basic protein of 192 aa. Vif has been shown to be essential for the modulation of virion infectivity in nom1al host cells and is believed to function by interacting with both viral and cellular proteins. More recent studies have focused on its involvement in controlling the encapsidation of cellular proteins ~f the APOBEC3 family. However earlier work of Vif suggested an involvement in viral morphogenesis and this was the main focus of the present study. Vifhas been shown to be associated with the viral nucleocapsid and to be specifically packed into HIV patiicles, either by interaction with viral RNA and/or Gag and GagPol precursors. This study had as its primary aim definition of the molecular interactions of Vif with the Gag precursor (Pr55GAG ), the viral Protease (RR), and the antiviral cellular proteins APOBEC3G/3F. An in vivo mammalian two-hybrid assay was used to study the interactions between Vif and both Pr55GAG and the viral PR. This found that Vif interacts with Pr55GAG • To begin mapping the positions of this interaction, a series of mutations were made in both proteins. Complimenting previous studies on Vif done at Warwick, amino acid 21 was found to be cmcial for the interaction between Vif and Pr55GAG in the mammalian two-hybrid assay. Interaction between Vif and Pr55GAG was fmiher confirmed using an independent in vitro GST pull-down assay. No interactions were found between Vif and PR and between PR and Pr55GAG • The second objective of this study was to analyse the molecular interactions of HIV-1 Vif, Pr55GAG , and PR with the APOBEC3 family of cellular proteins using both an in vivo mammalian two-hybrid assay and an in vitro GST pull-down assay. In the mammalian two-hybrid assay a direct interaction between Pr55GAG and both APOBEC3G and 3F was identified. These interactions were further confirmed using the GST pull-down assay. A direct interaction between Vif and the two APOBEC proteins APOBEC3G and 3F was also seen in vitro in the GST pull-down assay; however, these interactions could not be seen in vivo using the mammalian two hybrid assay. A third are~ of work was concemed with the high level expression of Vifin a bacterial expression system and purification of the protein for structural studies. This experimental work was complemented by computer based model building using a comparative modeling method. The aim of this work being to produce an atomic level resolution model for Vifwhich could be tested against the experimental results achieved in interaction site mapping studies. Building on earlier work done at Warwick a fourth area of this study involved in vivo experiments aimed at understanding the role of HIV-1 Vif iil resistance to protease inhibitors (Adekale et al., 2005). This involved establishing the molecular reagents to allow the generation of infectious molecular clones carrying various variants of Vifand the HIV-1 protease. Plasmid constructs were generated to allow the inse11ion of different variants of the PRgene into an infectious molecular clone building on the previously available strategy which allowed similar exchanges with the Vifgene.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Superior effect of combination vs. single steroid therapy in keloid disease: A comparative in vitro analysis of glucocorticoids

Keloid disease (KD) is a fibroproliferative disorder of unknown etiology. Current use of corticosteroid injection is partially beneficial with 80% recurrence rate. Additionally, the efficacy of different steroids, alone or in combination as opposed to monotherapy, in treating KD remains unclear. Here, we compared the single and combined efficacy of glucocorticoids - dexamethasone (Dex), triamcinolone (TAC), and methylprednisolone (Medrol) - on primary keloid fibroblasts (KFs) (n = 27) and normal skin (n = 19) fibroblasts at cellular, protein, and messenger RNA levels in vitro. Our results demonstrated that cytotoxicity to steroids was dose dependent. Cell spreading, attachment, and proliferation were significantly (p <0.05) reduced by Medrol and TAC. Migration and invasion properties of KF were inhibited significantly (p <0.05) by Medrol and TAC compared with Dex. At both protein and messenger RNA levels, keloid-associated fibrotic markers were significantly (p <0.05) decreased by Medrol and TAC compared with Dex. However, vascular endothelial growth factor expression was significantly (p = 0.01) decreased by Dex compared with TAC and Medrol. Medrol and TAC caused significant (p <0.04) apoptosis, whereas Dex inhibited the UV-induced apoptosis and up-regulated survivin. Blocking of glucocorticoid receptor by RU486 inhibited cytoprotective property of Dex and apoptotic properties of TAC and Medrol. Double treatment with Dex + TAC and Dex + Medrol significantly (p <0.05) induced apoptosis. In conclusion, this is the first study to report the efficacy of three well-known steroids on KF and suggest that combination may be superior than using a single steroid in treating KD. © 2012 by the Wound Healing Society

Notch signaling pathway in keloid disease: Enhanced fibroblast activity in a Jagged-1 peptide-dependent manner in lesional vs. extralesional fibroblasts

Keloid disease (KD) is a fibroproliferative disorder of unknown etiopathogenesis with ill-defined treatment. There is increasing evidence to suggest that aberrant Notch signaling may contribute directly to skin pathogenesis and altered expression of Notch receptors identified in KD. Therefore, the aim of this study was to investigate the Notch signaling pathway in KD compared to normal skin (NS). In this study, we employed in vitro primary cell culture models to elucidate the role of Notch signaling in 44 tissue samples from patients with KD split into keloid and extralesional (EL) samples (internal control) from the same patients, and six NS tissue samples (external control). We show the presence of a significant (p <0.05) up-regulation of Notch receptors and ligand Jagged-1 (JAG-1) in KD compared to EL and NS tissue samples. Cell spreading, attachment, and proliferation were significantly (p <0.05) reduced in JAG-1 antisense-treated primary dermal fibroblasts isolated from KD and treated with γ-secretase inhibitor (blocks proteolytic cleavage and activation of Notch), evaluated by real-time cell analyzer (RTCA) on a microelectronic sensory array. In contrast, extralesional skin fibroblasts (ELF) treated with recombinant human JAG-1 (rh-JAG-1) peptide showed significant (p <0.05) enhancement of cell spreading, attachment, and proliferation in RTCA. Activation/inhibition of JAG-1 and Notch signaling significantly (p <0.05) altered the behavior of primary keloid fibroblasts and ELF, in cell migration (using a scratch wound assay), invasion (using a 3D invasion assay), and angiogenesis (in vitro coculture tube formation assay). In conclusion, this is the first study to demonstrate a potential role for the Notch signaling pathway in KD progression and that targeting this pathway may provide a novel strategy for treatment of KD. © 2012 by the Wound Healing Society