Is gibbon ape leukaemia virus still a threat?

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Gene therapy for PIDs: Progress,pitfalls and prospects

Substantial progress has been made in the past decade in treating several primary immunodeficiency disorders (PIDs) with gene therapy. Current approaches are based on ex-vivo transfer of therapeutic transgene via viral vectors to patient-derived autologous hematopoietic stem cells (HSCs) followed by transplantation back to the patient with or without conditioning. The overall outcome from all the clinical trials targeting different PIDs has been extremely encouraging but not without caveats. Malignant outcomes from insertional mutagenesis have featured prominently in the adverse events associated with these trials and have warranted intense pre-clinical investigation into defining the tendencies of different viral vectors for genomic integration. Coupled with issues pertaining to transgene expression, the therapeutic landscape has undergone a paradigm shift in determining safety, stability and efficacy of gene therapy approaches. In this review, we aim to summarize the progress made in the gene therapy trials targeting ADA-SCID, SCID-X1, CGD and WAS, review the pitfalls, and outline the recent advancements which are expected to further enhance favourable risk benefit ratios for gene therapeutic approaches in the future.     

Oncoretroviral gene transfer to NOD/SCID repopulating cells using three different viral envelopes

Background

The aim of this study was to investigate gene transfer to human umbilical cord blood (CB) CD34⁺/CD38^low and NOD/SCID repopulating cells using oncoretroviral vectors and to compare the transduction efficiency using three different viral envelopes.

Methods

CB cells were transduced on Retronectin using an MSCV‐based vector with the gene for GFP (MGIN), which was packaged into three different cell lines giving different envelopes: PG13‐MGIN (GALV), 293GPG‐MGIN (VSV‐G) or AM12‐MGIN (amphotropic).

Results

Sorted CD34⁺/CD38^low cells were efficiently transduced after 3 days of cytokine stimulation and the percentage of GFP‐positive cells was 61.8±6.6% (PG13‐MGIN), 26.9±3.5% (293GPG‐MGIN), and 39.3±4.8% (AM12‐MGIN). For transplantation experiments, CD34⁺ cells were pre‐stimulated for 2 days before transduction on Retronectin preloaded with vector and with the addition of 1/10th volume of viral supernatant on day 3. On day 4, the expanded equivalent of 2.5×10⁵ cells was injected into irradiated NOD/SCID mice. All three pseudotypes transduced NOD/SCID repopulating cells (SRCs) equally well in the presence of serum, but engraftment was reduced when compared with freshly thawed cells. Simultaneous transduction with all three vector pseudotypes increased the gene transfer efficiency to SRCs but engraftment was significantly impaired. There were difficulties in producing amphotropic vectors at high titers in serum‐free medium and transduction of CD34⁺ cells using VSV‐G‐pseudotyped vectors under serum‐free conditions was very inefficient. In contrast, transduction with PG13‐MGIN under serum‐free conditions resulted in the maintenance of SRCs during transduction, high levels of engraftment (29.3±6.6%), and efficient gene transfer to SRCs (46.2±4.8%).

Conclusions

The best conditions for transduction and engraftment of CB SRCs were obtained with GALV‐pseudotyped vectors using serum‐free conditions. Copyright © 2002 John Wiley & Sons, Ltd.

     

Adenoviral vectors expressing fusogenic membrane glycoproteins activated via matrix metalloproteinase cleavable linkers have significant antitumor potential in the gene therapy of gliomas

BACKGROUND: Fusogenic membrane glycoproteins (FMG) such as the gibbon ape leukemia virus envelope (GALV) glycoprotein are potent therapeutic transgenes with potential utility in the gene therapy of gliomas. Transfection of glioma cell lines with FMG expression constructs results in fusion with massive syncytia formation followed by cytotoxic cell death. Nevertheless, ubiquitous expression of the GALV receptor, Pit-1, makes targeting desirable in order to increase the specificity of the observed cytopathic effect. Here we report on use of matrix metalloproteinase (MMP)-cleavable linkers to target the cytotoxicity of FMG-expressing adenoviral vectors against gliomas. METHODS: Replication-defective adenoviruses (Ad) were constructed expressing the hyperfusogenic version of the GALV glycoprotein linked to a blocking ligand (C-terminal extracellular domain of CD40 ligand) through either an MMP-cleavable linker (AdM40) or a non-cleavable linker (AdN40). Both viruses also co-expressed the green fluorescent protein (GFP) via an internal ribosomal entry site. RESULTS: The glioma cell lines U87, U118, and U251 characterized by zymography and MMP-2 activity assay as high, medium and low MMP expressors, respectively, the MMP-poor cell lines TE671 and normal human astrocytes were infected with AdM40 and AdN40 at different multiplicities of infection (MOIs) from 1-30. Fusion was quantitated by counting both number and size of syncytia. Infection of these cell lines with AdN40 did not result in fusion or cytotoxic cell death, despite the presence of infection, as demonstrated by GFP positivity, therefore indicating that the displayed CD40 ligand blocked GALV-induced fusion. Fusion was restored after infection of glioma cells with AdM40 at an MOI as low as 1 to an extent dependent on MMP expression and coxsackie adenovirus receptor (CAR) expression in the specific cell line. Western immunoblotting demonstrated the presence of the cleaved CD40 ligand in the supernatant of fused glioma cells. Use of the MMP inhibitors 1,10 phenanthroline and N-hydroxy-5,5-dimethylpiperazine-2-carboxamide completely abolished AdM40-induced fusion, while the non-specific serine protease inhibitor soybean trypsin inhibitor did not affect it, thus demonstrating specificity of the observed effect. Intratumoral treatment of BalbC/nude mice bearing subcutaneous U87 glioma xenografts with AdM40 at a total dose of 1.2 x 10(10) plaque-forming units (pfu) resulted in statistically significant tumor regression as compared with control animals either treated with AdN40 (p = 0.01) or untreated animals (p = 0.01). Treatment with AdM40 also resulted in survival improvement as compared with AdN40-treated animals (p = 0.006) or untreated animals (p = 0.001). Histopathologic examination of treated tumors demonstrated extensive syncytia formation. CONCLUSIONS: Our data indicate that AdM40, a replication-defective adenovirus expressing the GALV fusogenic glycoprotein, attached to a blocking ligand via an MMP-cleavable linker, can target the cytotoxicity of GALV in MMP-overexpressing glioma lines and xenografts, and maintain significant antitumor activity both in vitro and in vivo. Given the high frequency of MMP overexpression in gliomas, AdM40 represents a potentially promising agent in the gene therapy of these tumors.     

Lack of specificity of cell-surface protease targeting of a cytotoxic hyperfusogenic gibbon ape leukaemia virus envelope glycoprotein

Background

In a strategy termed “Protease Targeting”, retroviral vectors carrying an EGF infectivity‐blocking domain fused to the N‐terminus of the envelope SU via a MMP (matrix metalloproteinase)‐cleavable linker were successfully used to target gene delivery to EGF receptor‐(EGF‐R‐)positive tumour cells over‐expressing MMPs. In the current study, we aimed to investigate whether this strategy could be applied to (a) limit the cytotoxic activity of a hyperfusogenic GALV therapeutic gene, and (b) enhance the immune‐stimulatory properties of GALV via local, MMP‐mediated release human granulocyte‐macrophage colony stimulating factor (GM‐CSF).

Methods

We generated GALV envelope expression constructs displaying EGF or GM‐CSF blocking ligands at the N‐terminus of GALV envelope SU via a non‐cleavable, Factor Xa protease or MMP‐cleavable linker and investigated their cytotoxicity on MMP‐positive and negative cell lines.

Results

The unmodified hyperfusogenic GALV envelope was cytotoxic to all cell lines tested. The non‐cleavable linker GALV envelope constructs caused no cytotoxicity, demonstrating efficient inhibition by the displayed domains. Moderate activation of fusion of the protease‐cleavable linker constructs was observed in all cell lines, regardless of their level of MMP expression and of the specificity of the linker. High levels of the ‘blocking domain’ were detected in the cell supernatants due to dissociation of the surface unit (SU) from the transmembrane (TM) component of the GALV envelope glycoprotein TM.

Conclusions

Unlike protease targeting in the context of retroviral vectors, protease activation of the cytotoxicity of GALV envelope by cleavage of a fusion blocking ligand at the cell surface does not appear to be specifically mediated by cell‐surface MMPs. In addition, shedding of the SU‐fusion protein from the TM limits the general applicability of this strategy for cancer gene therapy. Specificity of cell‐cell fusion mediated by GALV envelope cannot be manipulated in the same fashion as virus‐cell fusion. Copyright © 2002 John Wiley & Sons, Ltd.