M de Alwis

Publications (18)71.05 Total impact

• Article: Stable rAAV-mediated transduction of rod and cone photoreceptors in the canine retina.

  J W B Bainbridge, A Mistry, F C Schlichtenbrede, A Smith, C Broderick, M De Alwis, A Georgiadis, P M Taylor, M Squires, C Sethi, D Charteris, A J Thrasher, D Sargan, R R Ali
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  ABSTRACT: Recombinant adeno-associated virus (rAAV) vectors are attractive candidates for the treatment of inherited and acquired retinal disease. Although rAAV vectors are well characterized in rodent models, a prerequisite to their clinical application in human patients is the thorough evaluation of their efficacy and safety in intermediate animal models. In this study, we describe rAAV-2-mediated expression of GFP reporter gene in retinal cells following local vector delivery in dogs. Subretinal delivery of rAAV.CMV.GFP was performed unilaterally in eight normal dogs from 6 weeks of age. The area of retinal transduction was maximized by the optimization of surgical techniques for subretinal vector delivery by pars-plana vitrectomy and the use of fine-gauge subretinal cannulae to create multiple retinotomies. rAAV-2 vectors mediated efficient stable reporter gene expression in photoreceptors and retinal pigment epithelial cells. We found efficient transduction of cone photoreceptors in addition to rods in both the canine retina and after subretinal vector delivery in another intermediate animal model, the feline retina. GFP expression in dogs was confined to the area of the retinal bleb and was sustained in cells at this site for at least 18 months. Electroretinography demonstrated a modest reduction in global rod-mediated retinal function following subretinal delivery of rAAV.CMV.GFP. Three of the eight animals developed delayed-onset intraocular inflammation, in two cases associated with a serum antibody response to GFP protein. We conclude that rAAV-2 vectors mediate efficient sustained transgene expression in rod and cone photoreceptors following subretinal delivery in this intermediate animal model. The possibility of adverse effects including intraocular immune responses and reduced retinal function requires further investigation prior to clinical applications in patients.
  Gene Therapy 09/2003; 10(16):1336-44. · 3.71 Impact Factor
• Article: Hypoxia-regulated transgene expression in experimental retinal and choroidal neovascularization.

  J W B Bainbridge, A Mistry, K Binley, M De Alwis, A J Thrasher, S Naylor, R R Ali
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  ABSTRACT: Recombinant AAV vectors mediate efficient and sustained transgene expression in retinal tissues and offer a powerful approach to the local, sustained delivery of angiostatic proteins for the treatment of ocular neovascular disorders. The application of such strategies may also require regulated gene expression to minimize the potential for unwanted adverse effects. In this study, we have evaluated the effect of a hypoxia-responsive element (HRE) on the kinetics of recombinant adeno-associated (rAAV)-mediated reporter gene expression in murine models of retinal and choroidal neovascularization. In murine ischaemia-induced retinal neovascularization, intravitreal delivery of rAAV.HRE.GFP results in reporter gene expression specifically at sites of vascular closure during the period of active neovascularization and not after vector delivery in normal controls. In murine laser-induced choroidal neovascularization, subretinal delivery of rAAV.HRE.GFP results in reporter gene expression at sites of active neovascularization but not elsewhere or after vector delivery in normal controls. HRE-driven gene expression offers an attractive strategy for the targeted and regulated delivery of angiostatic proteins to the retina in the management of neovascular disorders.
  Gene Therapy 07/2003; 10(12):1049-54. · 3.71 Impact Factor
• Article: Inhibition of retinal neovascularisation by gene transfer of soluble VEGF receptor sFlt-1.

  J W B Bainbridge, A Mistry, M De Alwis, E Paleolog, A Baker, A J Thrasher, R R Ali
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  ABSTRACT: Retinal angiogenesis is a central feature of the leading causes of blindness. Current treatments for these conditions are of limited efficacy and cause significant adverse effects. In this study, we evaluated the angiostatic effect of gene transfer of the soluble VEGF receptor sFlt-1 in a mouse model of ischaemia-induced retinal neovascularisation using adenovirus and adeno-associated virus (AAV) vectors. We induced proliferative retinopathy in mice by exposure to 75% oxygen from postnatal day 7 (p7) to p12 and injected intravitreally recombinant viral vectors expressing the reporter green fluorescent protein (GFP) or vectors expressing the VEGF inhibitor sFlt-1. Efficient adenovirus-mediated GFP expression was evident in cells of the corneal endothelium and iris pigment epithelium. AAV-mediated GFP expression was evident in ganglion cells and cells of the inner nuclear layer of the retina. Vector-mediated sFlt-1 expression was confirmed by ELISA of pooled homogenised whole eyes. Injection of either vector expressing sFlt-1 resulted in a reduction in the number of neovascular endothelial cells by 56% and 52% for adenovirus and AAV vectors, respectively (P < 0.05). Local gene transfer of sFlt-1 consistently inhibits experimental retinal neovascularisation by approximately 50% and offers a powerful novel approach to the clinical management of retinal neovascular disorders.
  Gene Therapy 03/2002; 9(5):320-6. · 3.71 Impact Factor
• Article: Gene replacement therapy in the retinal degeneration slow (rds) mouse: the effect on retinal degeneration following partial transduction of the retina.

  G M Sarra, C Stephens, M de Alwis, J W Bainbridge, A J Smith, A J Thrasher, R R Ali
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  ABSTRACT: The retinal degeneration slow (rds or Prph2(Rd2/Rd2)) mouse, a model of recessive retinitis pigmentosa, lacks a functional gene encoding peripherin 2. This membrane glycoprotein is required for the formation of photoreceptor outer segment discs. The striking feature of the rds mouse is the complete failure to develop outer segments. We have previously examined the short-term effect of gene replacement therapy using an adeno-associated (AAV) vector and demonstrated induction of outer segments and improvement of photoreceptor function. Here we have extended our analysis and have demonstrated that the potential for ultrastructural improvement is dependent upon the age at which animals are treated, but the effect of a single injection on photoreceptor ultrastructure may be long-term. However, there was no significant effect on photoreceptor cell loss, irrespective of the date of administration, despite the improvements in morphology and function. Our investigation excluded procedure-related damage, vector toxicity and immune responses as major factors which might counteract the benefits of photoreceptor restoration, but suggested that transgene over-expression is of significance. These findings suggest that successful gene therapy in patients with photoreceptor defects may ultimately depend upon intervention in early stages of disease and upon accurate control of transgene expression.
  Human Molecular Genetics 11/2001; 10(21):2353-61. · 7.64 Impact Factor
• Article: Efficient and selective AAV2-mediated gene transfer directed to human vascular endothelial cells.

  S A Nicklin, H Buening, K L Dishart, M de Alwis, A Girod, U Hacker, A J Thrasher, R R Ali, M Hallek, A H Baker
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  ABSTRACT: Gene therapy vectors based on adeno-associated virus-2 (AAV2) offer considerable promise for human gene therapy. Applications for AAV vectors are limited to tissues efficiently transduced by the vector due to its natural tropism, which is predominantly skeletal muscle, neurons, and hepatocytes. Tropism modification to elevate efficiency and/or selectivity to individual cell types would enhance the scope of AAV for disease therapies. The vascular endothelium is implicitly important in cardiovascular diseases and cancer, but is relatively poorly transduced by AAV vectors. We therefore genetically incorporated the peptide SIGYPLP, which targets endothelial cells (EC), into position I-587 of AAV capsids. SIGYPLP-modified AAV (AAVsig) showed enhanced transduction of human EC compared with AAV with a wild-type capsid (AAVwt), a phenotype independent of heparan sulphate proteoglycan (HSPG) binding. In contrast, AAVsig did not enhance transduction of primary human vascular smooth muscle cells or human hepatocytes, principal targets for AAV vectors in local or systemic gene delivery applications, respectively. Furthermore, infection of EC in the presence of bafilomycin A(2) indicated that intracellular trafficking of AAV particles was altered by targeting AAV by means of SIGYPLP. AAV vectors with enhanced tropism for EC will be useful for diverse gene therapeutics targeted at the vasculature.
  Molecular Therapy 10/2001; 4(3):174-81. · 6.87 Impact Factor
• Article: Optimization of recombinant adeno-associated virus production using an herpes simplex virus amplicon system.

  E Feudner, M de Alwis, A J Thrasher, R R Ali, S Fauser
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  ABSTRACT: A major limitation of adeno-associated virus (AAV) based vectors for clinical applications to date is the production of high-titer recombinant AAV vector stocks. Despite recent improvements, the amount of recombinant adeno-associated virus vectors (rAAV) particles produced per cell continues to be significantly lower than that of wild-type AAV. In this study, an HSV-based system for rAAV production was used to examine the influence of different parameters including transfection conditions (vector-to-packaging plasmid ratio, amount of total transfected DNA, cell confluency) and multiplicity of infection of herpes helper virus on the resulting titre of rAAV stocks. For herpes helper virus, time-course experiments were carried out to analyse the effect on rAAV yields up to 72 h postinfection and to determine the ideal harvesting time. Taken together, the optimized production scheme consistently yields more than 3x10(3) transducing units per producer cell.
  Journal of Virological Methods 09/2001; 96(2):97-105. · 2.01 Impact Factor
• Article: In vivo myocardial gene transfer: optimization, evaluation and direct comparison of gene transfer vectors.

  M J Wright, L M Wightman, C Lilley, M de Alwis, S L Hart, A Miller, R S Coffin, A Thrasher, D S Latchman, M S Marber
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  ABSTRACT: The purpose was to determine the relative efficiency, toxicity and duration of expression following gene delivery by intramyocardial injection of naked DNA, naked DNA complexed to cationic liposomes, naked DNA complexed to cationic liposomes with integrin-targetting peptide, recombinant (E1-/E3-) adenovirus, recombinant adeno-associated virus and recombinant (ICP27-) herpes simplex virus. All vectors incorporated a LacZ reporter driven by a promoter containing the hCMV-IE promoter/enhancer. Efficiency was scored by counting positive cells in five standard microscopic sections harvested from the left ventricular apex. Rabbit hearts (n = 100) were examined from 2 to 56 days after injection. Uncomplexed and complexed naked DNA were very inefficient with less than one positive cell visible per heart. The viral vectors all resulted in robust gene expression with adenovirus being the most efficient by at least one order of magnitude before 21 days. However, despite disparate titres, the efficiency beyond 21 days of adenovirus and adeno-associated virus were comparable. In contrast to adeno-associated virus, both adenovirus and herpes-simplex virus were associated with a marked inflammatory response. Despite reporter gene activity appearing only after 21 days, adeno-associated virus shows comparative promise as a myocardial gene delivery vector.
  Archiv für Kreislaufforschung 03/2001; 96(3):227-36. · 7.35 Impact Factor
• Article: Adeno-associated and herpes simplex viruses as vectors for gene transfer to the corneal endothelium.

  T Hudde, S A Rayner, M De Alwis, A J Thrasher, J Smith, R S Coffin, A J George, D F Larkin
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  ABSTRACT: We examined the efficacy and cytopathogenicity of adeno-associated (AAV) and herpes simplex viruses (HSV) as vectors for gene transfer to corneal endothelial cells (CECs). Recombinant AAV and HSV were examined for their ability to deliver a lacZ histochemical marker gene to whole-thickness rabbit and human corneas ex vivo. Transgene expression was detected with histochemistry and quantified by a colorimetric assay. Rabbit and human corneas transduced with AAV showed increasing numbers of cells expressing marker gene over a 3- to 4-week period. Using 2.5 x 10(6) or 1.5 x 10(7) infective units for rabbit and human corneal specimens, respectively, approximately 2% of CECs expressed the reporter gene. HSV (10(6) plaque-forming units/specimen) transduced approximately 5% of rabbit and human CECs but showed cytotoxicity. In contrast to the duration of recombinant AAV-mediated lacZ expression, recombinant HSV expression was maximal at day 1 and declined to low levels at day 7. AAV is a promising vector, but its usefulness for corneal transduction is currently limited by the technical difficulties preparing high titres. The HSV vector examined is efficient but needs further genetic modification to prolong transgene expression and reduce its toxicity.
  Cornea 06/2000; 19(3):369-73. · 1.73 Impact Factor
• Article: High-titer recombinant adeno-associated virus production from replicating amplicons and herpes vectors deleted for glycoprotein H.

  X Zhang, M De Alwis, S L Hart, F W Fitzke, S C Inglis, M E Boursnell, R J Levinsky, C Kinnon, R R Ali, A J Thrasher
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  ABSTRACT: Production of high-titer rAAV is essential for in vivo clinical application. One limiting factor may be the failure of existing systems to replicate the packaging genome in such a way that expression of Rep and Cap proteins is coordinately amplified. DISC-HSV (disabled single-cycle virus) is a genetically modified herpes simplex virus (HSV) that by deletion of glycoprotein H (gH) is infectious only if propagated in a complementing cell line. In this study, we have used DISC-HSV as a helper for rAAV replication, and have simulated to some extent the amplication of the rep and cap genomes seen in wtAAV infection by incorporating both these and vector sequences in HSV amplicons. Facilitated production of AAV Rep and Cap proteins translates into a considerably improved recovery of rAAV, which transduces cells of the neuroretina in vivo with high efficiency. The potential for contamination with infectious herpes particles is eliminated by the use of noncomplementing (gH-) cell lines to propagate the virus, and by standard purification methods. The use of DISC-HSV and herpes-derived amplicons for production of rAAV may be a useful strategy for future in vivo studies and for clinical application.
  Human Gene Therapy 11/1999; 10(15):2527-37. · 4.22 Impact Factor
• Source

  Available from: csic.es

  Article: Adeno-associated virus gene transfer to mouse retina.

  R R Ali, M B Reichel, M De Alwis, N Kanuga, C Kinnon, R J Levinsky, D M Hunt, S S Bhattacharya, A J Thrasher
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  ABSTRACT: Ocular gene transfer may provide a means for arresting the retinal degeneration characteristic of many inherited causes of blindness, including retinitis pigmentosa (RP). Previously, we have shown in immunodeficient animals that recombinant adeno-associated virus (rAAV) mediates transduction of photoreceptors as well as the retinal pigment epithelium (RPE) following subretinal injection. In this study we extend these observations and show that highly purified recombinant AAV vectors encoding the reporter gene LacZ transduce photoreceptors in an immunocompetent mouse strain following subretinal injection and efficiently transduce ganglion cells after intravitreal injection. Levels of transduction increase over time. Sublethal gamma-irradiation is shown to facilitate this process.
  Human Gene Therapy 02/1998; 9(1):81-6. · 4.22 Impact Factor
• Article: Gene therapy for primary immunodeficiency.

  A J Thrasher, J Goldman, M de Alwis, C D Porter, C Kinnon
  Biochemical Society Transactions 06/1997; 25(2):537-40. · 3.71 Impact Factor
• Article: Generation of recombinant adeno-associated virus (rAAV) from an adenoviral vector and functional reconstitution of the NADPH-oxidase.

  A J Thrasher, M de Alwis, C M Casimir, C Kinnon, K Page, J Lebkowski, A W Segal, R J Levinsky
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  ABSTRACT: The human parvovirus, adeno-associated virus-2 (AAV-2), has many attributes that recommend its use as a gene transfer vehicle, including a broad tissue tropism, the ability to integrate stably into the host genome, and efficient transduction of cells which proliferate slowly. However, application to human gene therapy is currently limited by existing methods for generation of recombinant AAV (rAAV), resulting in relatively low transducing titres. In an attempt to overcome some of these problems, we have developed a defective adenoviral vector which improves the efficiency of rAAV vector delivery to cells in which rAAV is propagated, and from which the rAAV genome can be efficiently rescued. A functional copy of the p47phox gene was successfully transferred to cell lines derived from patients with autosomal recessive chronic granulomatous disease (CGD) by rAAV recovered in this way, and function of the NADPH-oxidase was restored to levels which were stable for at least 8 weeks. This method for generation of rAAV, although still limited by the need for cotransfection of AAV Rep and Cap functions, may permit recovery of higher titre transducing stocks from cell lines in which these genes are stably incorporated, and significantly reduces the risk of contamination with wild-type adenovirus (wtAd).
  Gene Therapy 10/1995; 2(7):481-5. · 3.71 Impact Factor
• Source

  Available from: Colin Casimir

  Article: Functional reconstitution of the NADPH-oxidase by adeno-associated virus gene transfer.

  A J Thrasher, M de Alwis, C M Casimir, C Kinnon, K Page, J Lebkowski, A W Segal, R J Levinsky
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  ABSTRACT: Chronic granulomatous disease (CGD) comprises a heterogeneous group of inherited conditions characterized biochemically by disordered function of a unique multicomponent enzyme system present in phagocytic cells, the NADPH-oxidase. Clinically, it is characterized by recurrent bacterial and fungal infections that are relatively resistant to treatment by conventional means. Curative bone marrow transplantation has been successfully achieved in a small number of cases, but the wider application of this procedure is limited by availability of suitable donor material. Somatic gene therapy would overcome this problem, and several groups have now shown correction of the biochemical defect in hematopoietic cells by retrovirus-mediated gene transfer. However, the failure of the current generation of retroviral vectors to efficiently transduce quiescent cells greatly restricts their potential for gene transfer to pluripotent hematopoietic stem cells. Given these limitations, we have constructed vectors based on adeno-associated virus and used these to transfer a functional copy of the p47phox gene to immortalized B cells derived from patients with p47phox-deficient autosomal recessive CGD. We show stable expression of protein and restoration of NADPH-oxidase function in these cells in the absence of selection. Adeno-associated virus vectors may overcome some of the limitations of retroviral gene delivery systems and may therefore be a useful vehicle for curative gene therapy of CGD and other primary immunodeficiencies.
  Blood 08/1995; 86(2):761-5. · 9.90 Impact Factor
• Source

  Available from: Tracy Lester

  Article: Trisomy X in a female member of a family with X linked severe combined immunodeficiency: implications for carrier diagnosis.

  T Lester, M de Alwis, P A Clark, A M Jones, F Katz, R J Levinsky, C Kinnon
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  ABSTRACT: We describe a family affected by X linked severe combined immunodeficiency (SCIDX1) in which genetic prediction of carrier status was made using X chromosome inactivation studies together with limited genetic linkage analysis. Linkage studies in this family showed a confusing pattern of inheritance for the X chromosome. A female with a random pattern of X chromosome inactivation in her T cells appeared to have inherited an X chromosome with four recombinations within 10 cM. The odds of this happening in a single meiotic event make this an unlikely explanation. Data obtained from studying the X chromosomes of her two unaffected sons showed that this could be explained simply on the basis of her having inherited three alleles each of the relevant polymorphic DNA loci. We used fluorescent in situ hybridisation (FISH) to confirm that this person had inherited three complete X chromosomes. Thus, although the results from X chromosome inactivation analysis indicated that this subject was not a carrier of the affected chromosome, FISH and genetic linkage analysis showed clearly that the affected chromosome had been inherited. The implications of this finding for diagnosis of carrier status in this family and for other families with X linked inherited immunodeficiencies is discussed.
  Journal of Medical Genetics 10/1994; 31(9):717-20. · 6.36 Impact Factor
• Article: Carrier determination for X-linked agammaglobulinemia using X inactivation analysis of purified B cells.

  L A Alterman, M de Alwis, S Genet, R Lovering, H Middleton-Price, G Morgan, A Jones, S Malcolm, R J Levinsky, C Kinnon
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  ABSTRACT: We report the development of a relatively quick and simple method for the assessment of X inactivation status for carrier determination in families affected by X-linked agammaglobulinemia (XLA). This method utilises an immunomagnetic separation technique for B cell purification and a polymerase chain reaction (PCR) based assay for the determination of methylation status at the androgen receptor (AR) gene locus to assess whether X inactivation is random or non-random at this locus. We report the results we have obtained using this assay to investigate females known to be carriers of various X-linked immunodeficiency disorders. In addition, we investigated four females from different families affected by XLA, two of whom were of unknown carrier status, and we discuss the results obtained with this and other X-inactivation assays. A similar assay has recently been described by Allen et al. (1992) and applied to members of one family affected by XLA.
  Journal of Immunological Methods 12/1993; 166(1):111-6. · 2.20 Impact Factor
• Article: Physical and genetic mapping in the region of Xq12-21, which contains the locus for X-linked severe combined immunodeficiency.

  A Jones, T Morris, M de Alwis, S Malcolm, R J Levinsky, C Kinnon
  Immunodeficiency 02/1993; 4(1-4):259-62.
• Article: Inhibition of retinal neovascularisation by gene transfer of soluble VEGF receptor sFlt-1

  JWB Bainbridge, A Mistry, M de Alwis, E Paleolog, A Baker, AJ Thrasher, RR Ali
• Article: Efficient and selective AAV2-mediated gene transfer directed to human vascular endothelial cells

  SA Nicklin, H Buening, KL Dishart, M de Alwis, A Girod, U Hacker, AJ Thrasher, RR Ali, M Hallek, AH Baker