Abstract
(presenting)
Dept. of Clinical Pharmacology, University of Oxford
Use of siRNA molecules for therapeutic applications requires development of new and existing delivery systems, a process that would be greatly facilitated by a pharmacodynamic positive-readout mouse model. The purpose of such a system would be to determine the occurrence of spatially off-target effects in non-target tissues. We previously engineered an in vitro positive-readout reporter system based on the lac operon, in which binding of the lac repressor to lac operator sites within the promoter region of a luciferase reporter gene, suppresses expression until alleviated by siRNA targeting lac. Developing the system for the mouse model requires replacement of the viral promoters with ubiquitously expressed human promoters and modification of bacterial codons within lac to mammalian codons to prevent silencing. Such a system developed by Scrable et al. is currently being constructed and tested. Following pronuclear injection of fertilised oocytes, lines that express either luciferase, under the control of the Huntington's Disease promoter (embedded with lac operator sites), or mammalianised lac repressor, under the control of the β-actin promoter, ubiquitously throughout the mouse, were established. These lines were then crossed to generate double transgenics in which luciferase expression is repressed until re-established by administration of IPTG (altering the conformation of lac preventing it from binding to the operator sequences) in the drinking water. At the same time serum stable siRNA that specifically targets mammalianised lac was generated. We aim to use this model to investigate pharmacodynamics of various chemically modified siRNAs or siRNA delivery vehicles.
(presenting)
University of Manchester
Saphenous vein (SV) is the most frequently used conduit for coronary artery bypass grafting. Its long-term usefulness in this role is limited by progressive luminal loss, resulting from neointimal hyperplasia (NIH) and superimposed atherosclerosis. NIH is primarily associated with extracellular matrix (ECM) deposition on the luminal surface of the graft. There is substantial evidence to implicate TGF-β1 in NIH, and antagonists of TGF-β1 have been effective at inhibiting experimental NIH in injured arteries, but antagonism of TGF-β1 has not been shown to suppress NIH in vein grafts. We have investigated this possibility using a transgenic antagonist of TGF-β. The cDNA of the latency-associated peptide of TGF-β1 (LAP-β1) was isolated from full-length human TGF-β1 by PCR. After sequence confirmation the cDNA was incorporated into Ad5-LAP-β1. Its ability to antagonize TGF-β was confirmed in vitro before gene transfer into surgically-prepared SV. Vein segments were exposed to 108 pfu of Ad5-LAP-β1 or Ad5-lacZ then incised longitudinally and cultured for 14 days. Thereafter, segments were fixed, paraffin-embedded, sectioned and stained for elastic tissues before assessment by computerised morphometry. The neointimal area, maximum neointimal thickness and neointima/media ratio were reduced by 59% ( p < 0.0004), 53% ( p < 0.007) and 63% ( p < 0.0001) respectively in vein segments exposed to Ad5-LAP-β1 by comparison with segments infected with Ad5-lacZ. LAP-β1 is a potent antagonist of TGF-β1, abolishing TGF-β1 signalling in vitro, and suppresses NIH formation in surgically-prepared SV segments. This is the first time that a specific antagonist of TGF-β has been shown to be effective in this setting.
(presenting)
UCL , Windeyer Building, 46 Cleveland Street, London, W1T 2AH
Retroviral vectors are promising tools for gene therapy, but they can be oncogenic. The development of safer vectors depends on a quantitative assay for insertional mutagenesis and therefore we have established an in vitro assay (Bokhoven J Virol 2009, 83:283–294). Isolation of factor independent mutants from the IL-3 dependent cell line Bcl-15 provides a rapid, reproducible and cost-effective method of determining the safety of vectors. The lentiviral vector HV, with intact HIV LTRs, an internal SFFV (spleen focus forming virus) enhancer/promoter and a WPRE (woodchuck hepatitis virus posttranscriptional regulatory element), generated factor independent mutants. These mutants all have insertions in intron 1 of the Ghr (growth hormone receptor gene) and express a fusion mRNA transcript, which is initiated from the 5′ LTR of the vector and splices from the HIV splice donor site to the second exon, the first coding exon. This induces presentation of GHR on the cell surface and bovine growth hormone in the culture medium stimulates cell proliferation via GHR. We have recently extended our study to lentiviral vectors developed for clinical use, including a SIN (self-inactivating) lentiviral vector expressing the cytokine common gamma chain. This vector has a UCOE (ubiquitously acting chromatin opening element) house-keeping gene promoter, human A2UCOE (Zhang Blood 2007, 110:1448–57), and in our assay, factor independent mutants were obtained through transcriptional activation of the Ghr. In contrast to the HV mutants, transcription was initiated from within the UCOE element and spliced from a UCOE splice donor site to the second exon of Ghr.
(presenting)
Institute of Ophthalmology , 11-43 Bath Street, London, EC1V 9EL
We aim to assess the efficiency and kinetics of retinal pigment epithelium (RPE) specific gene knockdown using lentiviral short hairpin (shRNA) expression cassettes. Epithelial cell cycle modulators, ZO-1 and ZONAB, are part of an epithelial tight junction pathway cascade that regulates gene expression and RPE differentiation. Modulation of junctional components in epithelial cells is known to cause transformation towards a mesenchymal fate in vitro (epithelial-mesenchymal transition (EMT)) through loss of contact inhibition but this hypothesis has not been tested in vivo. Short hairpin RNA molecules targeting ZO-1, ZONAB and eGFP were cloned into a lentiviral backbone driven by a U6 promoter. We analysed the eyes at 5 and 10 day timepoints by light and electron microscopy, BrdU incorporation, as well as immunohistochemistry for ZO-1, ZONAB, RPE65 and RT-PCR for EMT markers from laser microdissected cells. Robust phenotypic changes are apparent 5 days after injection at a low titre. Downregulation of ZO-1 leads to EMT and RPE proliferation and migration. Downregulation of ZONAB leads to reduced cell density marked by downregulation of RPE65, an abundant RPE-specific gene, as well as onset of EMT marker upregulation through loss of contact inhibition. Our data suggest that as early as 5 days after subretinal delivery the transcribed shRNA reaches physiologically significant concentrations in the transduced cells. While establishing efficient RPE gene silencing that would be needed to treat RPE-specific dominant disorders, we also demonstrate the importance of junctional signalling in RPE proliferation and EMT through modulation of the ZO-1/ZONAB pathway.
(presenting)
Molecular Immunology Unit, Institute of Child Health London
UK
Adenosine deaminase (ADA) deficiency leads to lymphoid lineage toxicity resulting in severe combined immunodeficiency (SCID). Furthermore, the lack of this ubiquitously expressed enzyme causes accumulation of toxic products throughout the body, leading to hepatic, skeletal and neurological abnormalities in addition to the immunodeficiency. ADA-SCID patients treated using conventional gammaretroviral vectors show long-term immunological recovery and some metabolic correction; however, for the treatment of the non-immunological disease manifestations, improved systemic detoxification may be advantageous. Our strategy is to enhance the expression of the therapeutic gene in the erythrocyte lineage, whilst maintaining the expression levels in the lymphocyte populations. To achieve this, we have designed a lentiviral construct where GFP expression is driven by the house keeping gene EF1a promoter, linked to a minimal version of the human β-globin locus control region (βLCR). In cell lines, as well as in an in vitro model of human erythrocytes, this combination is capable of enhancing the expression of GFP by three-fold, in comparison to a control vector with only the EF1a promoter. In a mouse model of bone marrow transplant, the LCR-promoter is capable of enhancing the transgene expression in the erythrocyte lineage by 20-fold, without compromising the expression in other lineages. Our LCR-promoter combination significantly improves erythroid expression, and now we plan to apply this strategy to a vector expressing ADA, in order to correct a murine model of ADA deficiency.
(presenting)
Department of Biological Sciences, University of Warwick , Coventry, UK
Adenoviruses (Ads) have many attractive characteristics for use as agents of gene-based vaccines and therapies. The most popular Ad vector in preclinical research is based on Ad5. However, in the clinical setting Ad5 vectors have severe limitations. About 90% of the population have neutralising antibodies against Ad5 and infection requires the expression of the viral receptor CAR, which is not expressed on important cell types. Previous data from our lab suggested that the species D adenovirus, Ad19a, may overcome some of these limitations. Moreover, its highly efficient infection of human dendritic cells, which are critical for antigen presentation, may make the Ad19a vector suitable for vaccination. To investigate its potential for vaccination, two model antigens, the nucleocapsid gene from pneumovirus of mice (PVM-N), and the HIVA polyprotein cassette developed by Hanke, were inserted into replication-deficient Ad5 and Ad19a vectors (rAd5PVM, rAd19aPVM, rAd5HIVA & rAd19aHIVA). As previously noted for Ad19aGFP, rAd19aPVM and rAd19aHIVA expressed a significantly higher amount of transgene as compared to their Ad5 counterparts. We subsequently examined the ability of rAd5PVM and rAd19aPVM to protect mice from lethal challenge with PVM by using various intranasal prime/boost vaccinations. rAd19aPVM but not rAd5PVM protected mice at a dose of 106 pfu. However, rAd5PVM did protect mice at this dose when given with rAd19aPVM in a heterologous prime/boost schedule. Protection did not correlate with vaccination-induced IgG responses to PVM-N, implicating cell-mediated immune responses for protection. Furthermore, data will be presented on the elusive Ad19a receptor utilising rAd19aGFP in a whole virus system.
(presenting)
Dept. of Hepatology & Transplantation, King's College London School of Medicine
Critical aspects of surgical technique and clinical applicability of hydrodynamic gene delivery to the liver remain unresolved. Crucially, there has not been any report of high levels of reporter gene expression except in rodents. The vascular anatomy of the rat liver permits selective hydrodynamic delivery to the left lateral lobe. We demonstrate that delivery of 1.5 ml at 25 ml/min via the terminal part of the portal vein, with outflow obstruction, gives excellent reporter gene expression (∼106 RLU/mg). Without outflow obstruction, there is essentially no gene expression. The “minimally invasive” technique involves introducing a perfusion catheter into the right internal jugular vein, and then manipulating the catheter into a hepatic vein branch. It was used in the only published clinical study, and represents regional delivery without outflow obstruction. We demonstrate, in the pig model, that this technique (200 ml, 50 ml/sec to ∼20% of liver in 28-38 kg pigs) does not pressurise the targeted liver segment. We have developed a novel technique for regional hydrodynamic delivery to the right median lobe via the portal vein branch, with selective obstruction of the right hepatic vein. This technique maintains portal venous flow and results in excellent pressurisation of the right median lobe (200 ml, 50 ml/sec in 30-35 kg pigs). This demonstrates, for the first time, that pressurisation of the targeted lobe using a regional approach is possible. We conclude that outflow obstruction is essential for regional hydrodynamic gene delivery to the liver, and that it will therefore require open abdominal surgery.
(presenting)
British Heart Foundation Glasgow Cardiovascular Research Centre, University of Glasgow , 126 University Place, Glasgow G12 8TA, UK
One of the major limitations for adenoviral gene transfer in vivo is the profound liver tropism of adenovirus type 5 (Ad5). Recently, we demonstrated that coagulation factor X (FX) binds to Ad5-hexon protein at high affinity (1.95 nM) and this interaction mediates hepatocyte transduction following intravascular delivery. We developed novel Ad5 vectors genetically ablated for FX-interaction by modifying hexon hyper-variable region (HVR)5 and/or HVR7 and demonstrated significantly reduced hepatocyte transduction following intravenous administration. Here, we report detailed biodistribution and tropism of FX-binding ablated vectors at increasing doses (1 × 1010 to 1 × 1011 vp/mouse) in the presence or absence of macrophage depletion. Following intravascular delivery, FX-binding ablated Ad5 were predominantly localised in the liver and spleen after 1h. However, all FX-binding ablated Ad5 had substantially reduced hepatocyte transduction at 48h, in both control and macrophage-depleted mice. At high doses, and only in macrophage-depleted mice, FX-binding ablated vectors showed higher levels of splenic transduction compared to Ad5. Transgene localisation was observed in the marginal zone around the white pulp in the spleen. Systemic cytokine and chemokine profiles were not different between FX-binding ablated Ad5 and control Ad5 at low and intermediate doses, although higher levels of circulating interleukin (IL)-12, monokine induced by gamma interferon (MIG) and interferon-gamma-induced protein (IP-10) ( p < 0.05 vs Ad5) were observed in mice injected with the highest dose of FX-binding ablated Ad5 in both control and macrophage depleted mice. In summary, FX-binding ablated vectors show favorable biodistribution profiles but mediate enhanced splenic transduction under conditions of macrophage depletion at high doses.
(presenting)
Experimental Fetal Medicine Group, Department of Obstetrics and Gynaecology, National University of Singapore , Singapore
Long-term safety and efficacy of intrauterine gene transfer (IUGT) must be assessed before treating monogeneic conditions that cause irreparable fetal damage. We critically evaluate outcomes following IUGT with scAAV-FIX in a non-human primate model. Thirteen macaque fetuses received 4 × 1012 vg/fetus of scAAV2/8- or scAAV2/5-LP1-hFIXco delivered via the fetal intrahepatic vein at 0.9G. Plasma hFIX levels were serially measured. Liver biopsies were performed six-monthly for transduction efficiency and transgene expression. Maternal viraemia (2-1200 vg/ml) was detected up to 72 hours post-injection. Low-level maternal tissue transduction was detected. Maternal anti-AAV5 antibody response lasted beyond 15 months; anti-AAV8 antibodies resolved within 2-5 months. A brisk anti-AAV reaction in offspring did not nullify transgene expression. Offspring plasma hFIX demonstrated an initial decline, reaching trough levels at 30–60 days postnatal. These levels were then maintained stably. With AAV8, hFIX remained at ∼100% of normal activity from 2–11 months. With AAV5, hFIX ranged from 5–30% in three offspring with one animal expressing hFIX at < 1% of normal activity. Both pseudotypes effectively transduced hepatocytes with no difference in mean cell vector copies. AAV5 lead to significantly higher vector copies per cell compared to AAV8 in the cerebellum, spleen, adrenal gland and lung. FISH suggested persistence of AAV genome and LAM-PCR analysis revealed a highly polyclonal amplicon pattern. Identification of integrated and episomal vector forms is ongoing. Our data thus demonstrates persistent transgene expression arising from a single injection of scAAV in late gestation, which, in the context of expanding blood and liver volumes, raises the possibility of integration events.
(presenting)
Centre for Rheumatology, University College London
Regulatory T cells (Tregs) have the ability to control a wide range of immune responses, making them ideal candidates for the treatment of immune-mediated diseases. Indeed, in murine models of immunopathology adoptive therapy with antigen-specific Tregs, from T cell receptor (TCR)-transgenic mice, has shown considerable efficacy. However, translation of these studies into the clinic has been impeded by difficulties in identifying and isolating sufficient antigen-specific Tregs. We have recently proposed two novel gene transfer strategies to reliably generate large populations of antigen-specific Tregs. Firstly, using retroviral vectors we directly transduced a sorted Treg population with a TCR of known specificity: and secondly, we converted conventional CD4+ T cells by co-transduction of TCR along with the regulatory transcription factor FoxP3. Both approaches generated large populations of antigen-specific Tregs capable of efficient suppression in vitro. Furthermore, when transferred into recipient mice these cells engrafted efficiently and retained transgene expression. In an established model of arthritis we demonstrated antigen-driven accumulation of the gene modified T cells at the site of inflammation, which corresponded to a local reduction in the number of inflammatory Th17 cells and a significant decrease in arthritic bone destruction. Importantly, the specificity of the gene-modified Tregs was distinct from that of the arthritis-inducing T cells. In conclusion, we have demonstrated that adoptive therapy with gene-modified Tregs results in local, Ag-dependent control of immuno-pathology in the absence of systemic immune suppression. Furthermore, this work has highlighted the potential of Tregs to treat immunopathology via linked suppression without prior knowledge of the disease-initiating antigen.
(presenting)
Division of Infection and Immunity, University College of London ; Windeyer Institute, 46 Cleveland Street, London W1T 4JF, UK
Several pathways in antigen presenting cells (APC) are involved in the modulation of the immune responses to specific antigens. There is evidence that links inhibition of the ERK pathway to enhancement of the immunity, whilst its activation has been indirectly associated with immune suppression and generation of tolerance. Here we manipulate this pathway in dendritic cells (DC) using lentiviral vectors encoding constitutively-active or dominant-negative mutants of MEK1 and a model antigen gene. We demonstrate that simultaneous delivery of antigen and ERK activation in DC induces antigen-specific immune suppression, while ERK inhibition enhances the immune response. This is controlled by modulation in the surface expression of co-stimulatory/inhibitory molecules and is dependent on TGF-β. Furthermore, ERK activation induces differentiation of antigen-specific CD4+ Foxp3+ regulatory T cells with suppressive activity. These mechanisms can effectively inhibit inflammation in an arthritis model. This shows that ERK is a major regulatory pathway in APC involved in the induction of peripheral tolerance.
(presenting)
Infection and Immunity, University College London
A cross-strain T-cell vaccine against influenza which reduces the duration and severity of disease must expand large and polyfunctional CD4 and CD8 T-cell populations. Lentiviral transduction of dendritic cells (DC) with antigen together with downstream activators of DC signalling pathways is a potent means of achieving this. We have recently demonstrated dramatic up-regulation of costimulatory molecules CD80 and CD86 and increased IL-12 secretion by DC transfected with lentiviral vectors (LV) encoding viral FLICE-like inhibitory protein (vFLIP), a potent downstream activator of the NF-kappa B pathway. In this study we demonstrate that subcutaneous vaccination with LV expressing both vFLIP and influenza A NP (strain X31) induce potent IFN-positive CD8 T-cell responses which are superior to vaccination with LV expressing NP alone. LV encoding vFLIP and NP also skew the CD4 response towards a T-helper 1 phenotype. In a severe model of flu infection (influenza strain PR8/34 in balb/c), a single vaccination with 25 ng LV encoding vFLIP and NP 2-weeks prior to challenge (2 × LD50)conferred 40% protection compared with 0% protection in groups vaccinated with LV expressing NP alone (or saline, n = 10 per group). We are currently investigating means of further enhancing protection through both prime-boost regimens and also intranasal vaccination to enrich mucosa-associated CD8 T-cell memory populations.
(presenting)
British Heart Foundation Glasgow Cardiovascular Research Centre, Faculty of Medicine, University of Glasgow G12 8TA, UK
The production of defined cell lineages from human embryonic stem cells (hESC) is a critical requirement to evaluate their potential in regenerative medicine and basic scientific research. In cardiovascular medicine, tissue ischemia due to compromised blood flow is a major clinical burden and a substantial target for biological therapies, including gene and cell therapy. We sought to develop a defined feeder-free and serum-free directed differentiation protocol for the rapid generation of functional vascular endothelial cells (ECs) derived from hESC and to assess the system for microRNA regulation and angiogenesis. Directed differentiation revealed rapid loss of pluripotency markers and induction of vascular and angiogenesis-associated markers, assessed by mRNA, miRNAs and protein analysis concomitant with induction of EC morphology, assessed at 4, 7, 10, 14 and 21d post-induction of differentiation. We observed an induction in levels of angiogenic growth factors particularly at later differentiation time points. In vitro, hESC-ECs produced nitric oxide, migrated across a denuded wound and formed tubular network structures. In vivo, we report that 10d hESC-ECs (injected in the adductor muscle of immunodeficient mice subjected to hindlimb ischemia) incorporated into the vasculature and were able to induce therapeutic neovascularization. We report a highly efficient and scalable process for the differentiation of functional ECs from hESCs. This system may provide a renewable source of ECs for potential applications such as cellular therapy for the amelioration of regional ischemic tissue or for in vitro assays to assist in the search for novel gene products to develop new therapeutic approaches for vascular regeneration.
(presenting)
British Heart Foundation Glasgow Cardiovascular Research Centre, University of Glasgow , UK
Human immunodeficiency virus (HIV)-based lentivirus vectors (LV), have recently been utilised for ectopic gene transfer-mediated somatic cell reprogramming to induced pluripotent stem (iPS) cells. To date, no evidence exists suggesting multiple integration sites contribute to retroviral-mediated reprogramming. However, no association with integration site and LV-mediated reprogramming has been reported. Therefore, the biological relevance of LV integration site preferences remains unknown in iPS cell generation and remains an important safety consideration for potential clinical translational. We transduced normal human fibroblast cultures with self-inactivating (SIN) LV expressing green fluorescent protein (GFP) under control of the spleen focus forming virus promoter at an MOI of 200. 14–21d post-infection, colonies of cells with pluripotent morphology appeared in transduced cultures but not in parallel cultures lacking LV gene transfer. Cells were maintained in human embryonic stem cell (hESC) conditions. GFP +ve colony-derived cells (denoted LV-iPS cells) demonstrated ability to self-renew, expressed a mRNA and miRNA expression profile similar to pluripotent hESCs and bona fide iPS cells (but with a subset of LV-iPS-specific miRNAs), and formed embryoid bodies. Karyotyping revealed a broad range of genetic abnormalities. Comprehensive genome-wide integration site analysis revealed 23 insertion sites (preference for insertion in RefSeq genes), with altered mRNA levels observed in 60% genes located close to vector integrants selected for further analysis. We report induction of pluripotency in the complete absence of ectopic expression of iPS-reprogramming factors but with considerable transcriptional changes and insertional mutagenesis-associated detrimental changes in genomic structure and integrity induced by LV-mediated GFP gene transfer.
(presenting)
Nanomedicine Laboratory, Centre for Drug Delivery Research, The School of Pharmacy, Univeristy of London , London WC1N 1AX, United Kingdom
Gene therapy involves the delivery of a functional gene by a vector into target cells, resulting in a desired therapeutic effect. Adenovirus (Ad) has shown a great promise in gene therapy. However, in vivo studies have reported immunogenic responses and significant hepatotoxicity in liver. Such limitations have been overcome by engineering an artificially enveloped Ad using zwitterionic and cationic lipid formulations. However, this resulted in a significant reduction of gene expression due to the poor release of the lipid bilayer enveloped virus from the endosomal compartment. In the present work, we have explored the use of pH-sensitive DOPE:CHEMS lipid-envelope to enhance the virus release from the endosome following endocytosis. The surface engineered Ad were characterised by transmission electron microscopy (TEM), atomic force microscopy (AFM), dot blot, dynamic light scattering and zeta potential measurements. The gene expression of recombinant Ad encoding for beta-galactosidase reporter gene enveloped in DOPE:CHEMS showed high levels of gene expression when tested in different cell lines, comparable to naked Ad. These results were further confirmed by studying the intracellular trafficking of fluorescently-labelled, Cy3-Ad using confocal laser scanning microscopy. Interestingly, Cy-3 Ad enveloped in DOPE:CHEMS showed a uniform fluorescence distribution within the cytoplasm indicating Ad endosomal release. In addition, pH-sensitive enveloped Ad injected directly into human cervical adenocarinoma (C33a) xenografts grown on the flank of nude mice showed same level of gene expression to naked Ad. In conclusion, the artificially-enveloped Ad offers a promising tool in gene delivery since high level of Ad gene expression can be maintained with improved Ad immunogenicity in vivo.
(presenting)
Dept. Medical Oncology, Erasmus MC, Rotterdam, The Netherlands
Adoptive transfer of immune effector cells that are gene modified by retroviral transduction to express tumor-specific receptors constitute an attractive approach to treat cancer. In patients with metastatic renal cell carcinoma (RCC), we performed a study with autologous T-cells genetically retargeted with a chimeric antibody receptor (CAR) directed towards carbonic anhydrase IX (CAIX), an antigen highly expressed in RCC. In seven evaluable patients, we documented robust cellular and humoral anti-CAIX-CAR immune responses. In all cases, cellular immunity was directed against epitopes present in the complementarity-determining region (CDR) and framework region (FR) of the CAR-variable domains and occurred in the co-presence of an anti-idiotypic humoral response. The anti-idiotypic antibodies were able to neutralize CAIX-CAR-mediated T-cell function. These immune responses preceded a decreased peripheral persistence of functional CAIX-CAR T-cells and thereby are likely to compromise their anti-tumor efficacy. These observations may constitute a critical concern for CAR-retargeted T-cell therapy and underscore the need to attenuate immunogenicity of this approach.
(presenting)
Molecular Immunology Unit, Institute of Child Health , 30 Guilford Street, London, WC1N 1EH
Haemophilia A is a compelling candidate for treatment with gene therapy as therapeutic benefit only requires a modest increase in the endogenous coagulation factor level, response to treatment can be easily monitored, and FVIII expression can be mediated by many cell types in vivo. B-domain deleted (BDD) FVIII protein retains full procoagulant function and is expressed at higher levels than wild type FVIII. However a partial deletion of the B-domain leaving an N-terminal 226 amino acid stretch (N6) has been previously reported to increase in vitro secretion of FVIII tenfold compared to BDD-FVIII. In this study we tested FVIII variants with various B-domain constructs in the context of the wild-type or codon optimised FVIII coding sequence. The constructs were all expressed from a SIN lentiviral vector under control of the SFFV promoter in vitro in 293T cells, and in vivo after neonatal injection into FVIII knock-out mice. All constructs directed expression of detectable FVIII antigen and activity in vitro with the various B-domain constructs expressing equally well. The most striking difference was the effect of codon optimisation which led to a 16- to 20-fold increase in FVIII activity in vitro. Haemophilic mice treated with the viruses showed long-term expression of FVIII activity and phenotypic correction that was sustained for over 250 days. Addition of B-domain sequences to the BDD-FVIII did not significantly increase expression, however, expression from codon optimised constructs achieved levels of over 300% of normal human FVIII levels.
(presenting)
Molecular Immunology, Institute of Child Health , UCL, UK
Although anti-retroviral therapy is an effective treatment against HIV-1, the associated side effects are driving research for a long term cure. Genetic modification of haematopoietic stem cells (HSC) and/or peripheral blood lymphocytes to confer resistance against HIV-1 are being developed and some are currently at early phase clinical testing. In Owl Monkeys a strong inhibitor of HIV-1 has arisen by the retrotransposition of Cyclophilin A (CypA) DNA into exon 7 of the TRIM5 gene. This has resulted in a TRIM-Cyclophilin fusion protein with the B30.2 domain of TRIM5a replaced by CypA. We have designed humanised TRIM5 and TRIM21-CypA fusion variants with different length intergenic linker regions. HIV-1 derived vectors encoding SFFV and eGFP were used to deliver the factors to cell lines and human primary T cells. High titre vector stocks were produced and transduction was confirmed by flow cytometry. Protein-based analysis confirmed TRIMCyp expression in transduced cells. In cell lines and primary T cells transduced with the TRIMCyp vectors, entry of a HIV-1 vector expressing yellow fluorescent protein was reduced between 10 and 100 fold compared to untransduced cells or cells expressing eGFP alone. Cyclosporin treatment abrogated HIV inhibition, confirming the role of the CypA element. This data shows that these human mimics of the simian TRIMCyp restriction factors are effective inhibitors of HIV-1 infection. These factors could potentially be used as a gene therapy treatment to modify autologous HSC or T cells to provide a resistant pool of cells to repopulate the immune system.
(presenting)
MPS Stem Cell Research Group, Clinical Laboratory Sciences, University of Manchester , Manchester, UK
MPSIIIA is a lysosomal disorder caused by deficiency of the enzyme, sulfamidase (SGSH), that catabolises heparan sulphate (HS). Neither enzyme replacement therapy, nor haematopoietic stem cell transplant (HSCT) are able to correct the progressive neuropathy. We propose that this is due to a dose effect, where insufficient enzyme is produced by donor-derived brain microglial cells after HSCT. We have developed a stem cell gene therapy approach to overexpress SGSH and correct the disease in a MPSIIIA murine model. Lineage depleted bone marrow was transduced with an SGSH-lentiviral vector. 2.5 × 105 transduced or untransduced cells were administered to myeloablated MPSIIIA mice. Over 90% chimerism was standardly achieved. At 5 months post-HSCT with WT cells we find 42% of WT SGSH activity in PBS perfused spleens, 44% in livers and undetectable changes in activity in brains of MPSIIIA recipients. Recipients of SGSH-lentiviral transduced HSCT showed 160% of WT SGSH activity in spleen, 78% in liver and 10% in brain. Tandem mass spectrometry of HS oligosaccharides showed full normalisation of storage in liver and spleen and a 2.5 fold reduction of storage in brains of SGSH-lentiviral transduced HSCT MPSIIIA mice. This suggests that donor-derived microglia are present in the brain by 5 months and are secreting therapeutic levels of enzyme. In ongoing transplants a significant improvement in behaviour was observed in open field tests. The enzyme levels achieved, the reduction in HS oligosaccharides in the brain and the behavioural data suggest that this stem cell gene therapy approach may be viable for treating MPS IIIA.
(presenting)
NHLI, Imperial College , London
Sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) gene therapy improves mechanical function in heart failure, and is under evaluation in a clinical trial. A critical question is whether SERCA2a predisposes to increased SR leak by restoring SR Ca2+ levels?
(presenting)
Cell Therapy Group, School of Cancer and Enabling Sciences, University of Manchester
Engineering T cells with tumour specificity through the expression of targeting receptors has been shown to be practical with the approach currently being tested in the clinic. Two trials are underway in Manchester targeting Carcino-embryonic antigen (CEA) in gastro-intestinal cancer and CD19 in B cell lymphoma. Our preclinical studies have focused upon developing realistic models to investigate the therapeutic power of this approach against a background of natural expression of the target antigen and normal immune regulation. Mouse T cells targeting CEA can reject tumour in approximately 30% of C57 bl/6 mice but are effective in less than 6% of CEA transgenic mice where immune regulation to CEA is present. In the CD19 model, targeted T cells are highly efficient at eradicating long-term (13 day) established systemic A20 lymphoma and also cause a severe depletion of B cells expressing normal levels of the target antigen. However, B cells return and the engineered T cells disappear 45 days after transfer. In order to generate a more potent T cell response, we have engrafted mouse T cells with a more powerful receptor incorporating the CD28 signalling domain that results in long-term B cell depletion indicative of improved T cell function. However, with two particular constructs, a large proportion of treated animals suffered toxicity involving an apparent proliferation of CD4+ engineered T cells along with an expansion of host derived myeloid cells. The exact mechanism of toxicity is currently under investigation: however, these observations indicate the potential importance of autologous models to test therapies.
(presenting)
BHF GCRC, University of Glasgow , 126 University Place, Glasgow G12 8TA
Recent evidence suggests that adenovirus serotype 5 (Ad5) liver transduction after systemic administration is mediated by coagulation factor X (FX), which putatively bridges hexon proteins in the adenovirus capsid to heparan sulfate proteoglycans (HSPGs) at the cell surface. The aim of this study was to elucidate the specific HSPG requirements for FX-mediated transduction and to characterise the roles of coxsackie-adenovirus receptor (CAR) and av integrins during adenoviral trafficking via this recently-identified transduction pathway. Removal of HS sidechains by enzymatic digestion or competition with highly-sulfated heparins or heparan sulfates significantly decreased FX-mediated Ad5 transduction in vitro and ex vivo at the level of Ad5 binding. Removal of N-linked and, in particular, O-linked sulfate groups significantly attenuated the inhibitory capabilities of heparin, while the chemical inhibition of endogenous HSPG sulfation dose-dependently reduced FX-mediated Ad5 transduction. Unlike native heparin, modified heparins lacking O- or N-linked sulfate groups were unable to inhibit Ad5 accumulation in the liver 1h after intravascular administration. Similar results were observed in vitro using Ad5 vectors possessing mutations ablating CAR- and/or av integrin binding, demonstrating that attachment of the Ad5:FX complex to the cell surface is critically dependent on HSPG sulfation alone. Interestingly, Ad5 vectors ablated for av integrin binding showed markedly delayed trafficking kinetics, highlighting the need for an efficient post-attachment internalisation signal for optimal Ad5 uptake and trafficking via the FX transduction pathway. This study therefore integrates the established model of av integrin-dependent adenoviral transduction with the FX-mediated pathway underlying liver transduction by Ad5 following intravenous delivery.
(presenting)
BHF GCRC, University of Glasgow , 126 University Place, Glasgow, UK
Ad5-based vectors are undergoing development for clinical gene therapy and vaccine applications; however, a number of challenges remain to be overcome. A high affinity interaction between Ad5 hexon and coagulation factor-X mediates hepatocyte transduction. Additionally, the high seroprevalence of Ad5-neutralising antibodies in humans may limit its clinical efficacy. Ad48, a subgroup D adenovirus with low seroprevalence in humans, does not interact with FX. Substitution of the seven HVRs within the Ad5 hexon for those of Ad48, produced the hexon chimeric vector, Ad5HVR48(1-7) which failed to bind FX. In this study, we compared the in vivo pharmacokinetics of Ad5, Ad48 and Ad5HVR48(1-7) in macrophage-depleted, and untreated mice following intravenous injection of 3 × 1010 vp of each virus. Genome levels of Ad5, Ad48 and Ad5HVR48(1-7) in the livers of control animals did not differ 1 hr post-injection. Accumulation of Ad48, in both the liver and spleen, was sensitive to macrophage-depletion (∼23-fold and ∼13-fold reduction), whereas Ad5 and Ad5HVR48(1-7) sequestration was unaffected. In the livers of control animals 48hr post-injection, Ad5 and Ad5HVR48(1-7) genomes did not differ, however >65-fold lower levels of Ad48 genomes were detected. Macrophage-depletion increased the persistence of Ad5 and Ad48 genomes in the liver, but decreased Ad5HVR48(1-7) levels. In the spleen, Ad5 and Ad5HVR48(1-7) genomes were >28-fold higher than Ad48. Macrophage-depletion resulted in increased accumulation in the spleen with all viruses, notably Ad48. Inflammatory cytokines/chemokines were elevated in control and macrophage-depleted, Ad5HVR48(1-7)-treated animals. Selected inflammatory mediators were also elevated in Ad48-treated, control animals. These data suggest that large hexon modifications can lead to unpredictable biological outcomes.
(presenting)
Dept. of Genetics, UCL Institute of Ophthalmology
Department of Physiology, Anatomy and Genetics, University of Oxford
Familial Hypercholesterolaemia (FH) is a condition caused by mutations in the low density lipoprotein receptor (LDLR) gene. Expression of LDLR is regulated by intracellular sterols and over-expression leads to cytotoxic accumulation of cholesterol. We previously engineered vectors in which 10 kb of genomic DNA from the human LDLR locus encompassing the LDLR promoter drives expression of either the human LDLR cDNA or luciferase reporter (LDLR-Luc). We demonstrated that the promoter element confers stable, long-term, physiological regulation in vivo and functional complementation in vitro. We also demonstrated enhanced reporter gene expression in vivo following treatment with pravastatin. In this study we demonstrated that a more specific approach, targeting the mevalonate pathway with siRNA specific to HMG CoA reductase (HMGCR), led to enhanced transgene expression which may improve therapeutic outcome if used in conjunction with LDLR complementation gene therapy. In vitro we demonstrate that an 80% reduction in HMGCR protein in mouse and human cell lines resulted in enhanced expression from the LDLR-luc plasmid and an increase in endogenous LDLR expression. In vivo we performed hydrodynamic tail vein injections of the LDLR-Luc plasmid with siRNA specific for mouse Hmgcr and analysed luciferase expression up to one week post-injection using bioluminescence imaging. One week following injection we observed a thousand-fold higher level of luciferase expression in siRNA injected animals. This level of enhancement is two hundred times greater than that seen with pravastatin at the same time point. Our data suggest a major advantage of combining gene silencing with complementation gene therapy.
Department of Surgery, Division of Cardiovascular Surgery, The University of Pennsylvania Medical Center , Philadelphia, PA 19104, USA
Transgenic alteration of β-adrenergic signaling has produced new insight into the role of G protein-coupled receptor kinases (GRK) in heart failure (HF). GRK2/βARK1, specifically, has been implicated in the pathogenesis of dysfunctional cardiac βAR signaling and worsening HF. Murine studies have demonstrated βARKct as an effective in vivo inhibitor of βARK1 activity via Gβγ sequestration, highlighting a potential therapeutic target. This study's aim was to examine the effects of scAAV6-mediated βARKct on mechanoenergetics of normal cardiomyocytes using MCARDTM, a novel delivery platform. Specifically, we investigated βARKct's ability to increase BAR activity while preserving resting potential energy, the reverse found in HF patients. Six sheep were randomized to receive 1014 genome copies of scAAV6-βARKct, using MCARDTM, which provided for dual cardiac and systemic perfusion. Western blot/qPCR analysis confirmed robust βARKct density at euthanasia (12-16 wks). Using a 5F high-fidelity LV pressure transducer catheter, CINE cardiac MRI series were generated (t = pre-gene, 4 and 8 wks – post gene delivery). Systolic-diastolic pressure-volume area (PVA), potential energy per 100 gr (PE), myocardial oxygen consumption (MVO2), maximum elastance (Emax), end arterial elastance (Ea), and heart rate (HR) were evaluated at baseline and after isoproterenol stimulation. There were no significant differences in PVA, Emax, Ea and ejection fraction (EF) in control and βARKct-transfected hearts after 4 or 8 wks post infusion. Following isoproterenol infusion, however, the increase in CO from baseline was found significant in 8 weeks Δ692.2 ± 231.6 vs. Control Δ32.7 ± 176.7 ( p < 0.037) and PE loss decreased from 312.9 ± 92.8 to 49.7 ± 72.3J ( p < 0.026) [see Fig. 1]. HR increased across all time points ( p < 0.05). These results, supported with no load dependent variable interference, indicate that scAAV6-mediated MCARDTM delivery of βARKct to the normal heart significantly modified the BAR receptor response while preserving resting potential energy.
(presenting)
Institute for Women's Health, University College London , UK
The ability to efficiently deliver genes to the fetal brain represents a powerful research tool. A number of neonatal lethal neurodegenerative diseases present irreversible brain pathology during gestation. Therefore, neonatal intervention may be too late. Delivery of a therapeutic gene during gestation could provide answers to fundamental questions—e.g., Does early intervention increase life expectancy compared to neonatal intervention? What percentage of cells needs to be transduced to have an effect? Which neural cells should we be targeting? Recently, AAV2/9 was shown to cross the blood-brain barrier following neonatal administration. We have shown that in utero intravenous injection of both single-stranded (ss) and self-complimentary (sc) AAV2/9 expressing GFP into mice (embryonic day 15) results in efficient global transduction of neurons in the CNS. Furthermore, there is a stark contrast in cell type transduction when compared to neonatal administration and efficiency varied depending upon ss or sc configuration. Examination of injected mice by fluorescent microscopy, immunohistology, and GFP ELISA revealed extensive and efficient transduction in the visceral organs in addition to muscle, bone, eye, and skin. Gene expression was also seen in the peripheral nervous system such as the myenteric plexus of the gut. The combination of CNS and visceral organ transduction is well suited to the study of specific lysosomal storage diseases such as Type II Gaucher disease where both CNS and visceral pathology require targeting and a suitable mouse model is available.
(presenting)
University of Manchester
Disease of the pacemaker of the heart, the sinus node (SN), is common and often requires electronic pacemaker implantation. Biopacemaking, by expression of pacemaker channels in the heart, may offer an alternative therapy. A chimeric pacemaker channel (HCN212) induced rapid, but unstable pacemaking when expressed in dog ventricle. Repair of the SN may be superior to generation of a de novo ventricular biopacemaker. An ex vivo model of SN disease (SSS = sick sinus syndrome) was created by removal of the superior 2/3 of organ-cultured rat SNs. Pacemaking activity was monitored in intact SNs (n = 6), uninjected SSS preparations (n = 7) and SSS preparations injected with 2 × 107 pfu of Ad5-HCN212 (n = 9) or Ad5-GFP (n = 6). Recorded pacing activity was compared between groups by ANOVA. Unstable pacing was recorded in three Ad5-HCN212-injected preparations, but in no Ad5-GFP-infected or uninjected preparations. Control SSS preparations displayed significantly slower pacemaking than intact SNs ( p < 0.001). The mean pacing rate in the six Ad5-HCN212-infected preparations that exhibited stable pacing was significantly faster than that of the uninjected and Ad5-GFP-infected SSS preparations (both p < 0.001). These results confirm that adenovirus-mediated expression of HCN channels in a model of bradycardic SN disease can increase intrinsic pacing, and support the notion that modification of ion channel expression in the SN may confer therapeutic benefit in sinus node disease.
(presenting)
Queen Mary University of London , BJRU, WHRI
In this study we developed a secretable cell translocating NF-κB inhibitor that is produced and secreted by genetically engineered mammalian cells in sufficient amounts to enter and inhibit NF-κB activity in neighbouring cells. The secretable inhibitor LAP-TAT3-NBD consisted of three domains: the Latent Associated Peptide (LAP) from TGFβ which permits secretion from the producing cell, a protein transduction domain termed TAT3 and a small synthetic NF-κB inhibitor called Nemo Binding Domain (NBD). The ability of LAP-TAT3-NBD to inhibit NF-κB was tested in vitro using 57A HeLa cells which are permanently transfected so that the luciferase gene is expressed from an NF-κB regulated promoter. 57A HeLa cells that were treated with the secreted LAP-TAT3-NBD, demonstrated significantly reduced levels of luciferase activity after IL-1β stimulation confirming NF-κB inhibition. In subsequent studies we used a replication-deficient lentiviral vector either directly, or to engineer mouse fibroblasts to produce the secretable LAP-TAT3-NBD. The NF-κB inhibitory properties of the secreted LAP-TAT3-NBD were tested in vivo in a carrageenan-induced paw oedema model and in an antigen-induced arthritis model. Paws of mice treated with engineered cells or lentivirus encoding LAP-TAT3-NBD demonstrated significantly milder paw swelling, indicating that LAP-TAT3-NBD displays anti-inflammatory activity in vivo. These studies demonstrate that the LAP enables cell transducing molecules to be efficiently secreted from producing cells so they can have a therapeutic effect at an intracellular target in neighbouring cells. This methodology could be useful for the management of inflammatory diseases, such as rheumatoid arthritis and in other conditions where intracellular molecules are targeted.