Targeted retroviral gene delivery using ultrasound.

BACKGROUND: Achieving specificity of delivery represents a major problem limiting the clinical application of retroviral vectors for gene therapy, whilst lack of efficiency and longevity of gene expression limit non-viral techniques. Ultrasound and microbubble contrast agents can be used to effect plasmid DNA delivery. We therefore sought to evaluate the potential for ultrasound/microbubble-mediated retroviral gene delivery. METHODS: An envelope-deficient retroviral vector, inherently incapable of target cell entry, was combined with cationic microbubbles and added to target cells. The cells were exposed to pulsed 1 MHz ultrasound for 5 s and subsequently analysed for marker gene expression. The acoustic pressure profile of the ultrasound field, to which transduction efficiency was related, was determined using a needle hydrophone. RESULTS: Ultrasound-targeted gene delivery to a restricted area of cells was achieved using virus-loaded microbubbles. Gene delivery efficiency was up to 2% near the beam focus. Significant transduction was restricted to areas exposed to > or = 0.4 MPa peak-negative acoustic pressure, despite uniform application of the vector. An acoustic pressure-dependence was demonstrated that can be exploited for targeted retroviral transduction. The mechanism of entry likely involves membrane perturbation in the vicinity of oscillating microbubbles, facilitating fusion of the viral and cell membranes. CONCLUSIONS: We have established the basis of a novel retroviral vector technology incorporating favourable aspects of existing viral and non-viral gene delivery vectors. In particular, transduction can be controlled by means of ultrasound exposure. The technology is ideally suited to targeted delivery following systemic vector administration.

Complement insufficiency limits efficacy in a xenograft model of hyperacute rejection for cancer therapy.

Hyperacute rejection (HAR) is a rapid immunological response to an organ xenotransplant caused by recognition of endothelial galactose(alpha1,3)galactose (alphaGal) epitopes and complement-mediated cell lysis by host anti-alphaGal antibody ('natural antibody'). The alphaGal epitope is synthesised by a galactosyl transferase ((alpha1,3)GT) which humans lack. Because human cells transduced with (alpha1,3)GT are sensitised to natural antibody/complement-mediated lysis in human serum, delivery of (alpha1,3)GT to tumour vasculature in patients is a potential therapeutic strategy, by mimicking the pathophysiology of organ rejection. We therefore sought to develop an animal model of HAR for cancer therapy. Nude/(alpha1,3)GT knock-out mice allowed the growth of human tumour xenografts and the use of ecotropic retrovirus producer cells to generate expression of alphaGal on tumour vasculature. Lysis of alphaGal-positive murine endothelial cells with rabbit complement in conjunction with murine anti-alphaGal antibody in vitro was used to define the conditions necessary for HAR. However, tumour growth retardation and destruction of alphaGal-positive tumour endothelium were minimal after their administration, despite sera retaining post hoc cytolytic activity with fresh complement. The major limitation of this experimental system, of relevance to other therapeutic approaches, results from the use of a xenograft, in which additional xenoreactivities lead to complement insufficiency. Development of a tractable preclinical model in which to evaluate HAR for cancer therapy requires a syngeneic experimental system.

Spatial and acoustic pressure dependence of microbubble-mediated gene delivery targeted using focused ultrasound.

BACKGROUND: Ultrasound/microbubble-mediated gene delivery has the potential to be targeted to tissue deep in the body by directing the ultrasound beam following vector administration. Application of this technology would be minimally invasive and benefit from the widespread clinical experience of using ultrasound and microbubble contrast agents. In this study we evaluate the targeting ability and spatial distribution of gene delivery using focused ultrasound. METHODS: Using a custom-built exposure tank, Chinese hamster ovary cells in the presence of SonoVue microbubbles and plasmid encoding beta-galactosidase were exposed to ultrasound in the focal plane of a 1 MHz transducer. Gene delivery and cell viability were subsequently assessed. Characterisation of the acoustic field and high-resolution spatial analysis of transfection were used to examine the relationship between gene delivery efficiency and acoustic pressure. RESULTS: In contrast to that seen in the homogeneous field close to the transducer face, gene delivery in the focal plane was concentrated on the ultrasound beam axis. Above a minimum peak-to-peak value of 0.1 MPa, transfection efficiency increased as acoustic pressure increased towards the focus, reaching a maximum above 1 MPa. Delivery was microbubble-dependent and cell viability was maintained. CONCLUSIONS: Gene delivery can be targeted using focused ultrasound and microbubbles. Since delivery is dependent on acoustic pressure, the degree of targeting can be determined by appropriate transducer design to modify the ultrasound field. In contrast to other physical gene delivery approaches, the non-invasive targeting ability of ultrasound makes this technology an attractive option for clinical gene therapy.

Physical parameters affecting ultrasound/microbubble-mediated gene delivery efficiency in vitro.

Ultrasound (US)/microbubble-mediated gene delivery is a technology with many potential advantages suited to clinical application. Previous studies have demonstrated transfection but many are unsatisfactory in respect to the exposure apparatus, lack of definition of the US field or the limitations on parameters that can be explored using clinical diagnostic US machines. We investigated individual exposure parameters using a system minimising experimental artefacts and allowing control of many parameters of the US field. Using a 1-MHz transducer we systematically varied US parameters, the duration of exposure and the microbubble and DNA concentrations to optimise gene delivery. Delivery was achieved, using lipid microbubbles (SonoVue) and clinically acceptable US exposures, to adherent cells at efficiencies of approximately 4%. The acoustic pressure amplitude (0.25 MPa peak-negative), pulse repetition frequency (1-kHz) and duration of exposure (10 s) were important in optimising gene delivery with minimal impact on cell viability. These findings support the hypothesis that varying the physical parameters of US-mediated gene delivery has an affect on both efficiency and cell viability. These data are the first in terms of their thorough exploration of the US parameter space and will be the basis for more-informed approaches to developing clinical applications of this technology.

Primary attachment of murine leukaemia virus vector mediated by particle-associated heparan sulfate proteoglycan.

Target cell entry of murine leukaemia virus vectors proceeds via primary attachment, independent of the viral envelope protein and subsequent envelope-receptor interaction. Although much attention has been paid to modifying the latter for target cell specificity, the initial binding interaction has been overlooked, despite its opposing involvement both in providing the virus available for receptor binding and in depleting free virus. As a first step towards modifying primary attachment, both to provide specificity and to enhance vector availability, we sought to determine the nature of this interaction. Following an initial screen of GAGs (glycosaminoglycans) for their ability to inhibit virus binding and transduction, we have shown that production of virus from cells in which GAG sulfation is inhibited, or treatment of virus with heparinase III, reduces both particle attachment and infection. Detection in purified virus preparations of a neo-epitope generated by heparinase III confirmed the presence of virus-associated HSPG [HS (heparan sulfate) proteoglycan], acquired from the producer cell. We propose that host-acquired cell-surface HSPG (potentially including syndecan-2) provides a means of virus attachment to target cells that precedes specific receptor interaction and membrane fusion. Inhibition of HS biosynthesis may provide a sufficiently reduced background of primary binding such that novel mechanisms of attachment, ideally with appropriate target cell specificity, can be introduced.

Anti-alphaGal-dependent complement-mediated cytotoxicity in metastatic melanoma.

Antibodies to the cell surface disaccharide galactose(alpha1,3)galactose (alphaGal) are the most prevalent natural antibodies in human serum. The anti-alphaGal immunoglobulin M-dependent activation of complement causes hyperacute rejection of organ transplants from discordant species by human recipients. It has been shown in vitro that human tumour cells transduced with the gene that synthesizes alphaGal become sensitive to human serum. A prerequisite for anti-alphaGal antibody-based therapeutic strategies is that patients with cancer have adequate serum levels of anti-alphaGal immunoglobulins and complement. The objective of this work was to measure the levels and function of anti-alphaGal immunoglobulins and complement in the serum of patients with metastatic melanoma and healthy volunteers. Serum complement levels were assayed by radial immunodiffusion. Anti-alphaGal immunoglobulin G and immunoglobulin M titres were measured by enzyme-linked immunosorbent assay. Disaccharide sugar blocking was used to investigate antibody specificity. The functional integrity of anti-alphaGal antibodies and complement was investigated in cell lysis assays. It was found that the levels of the complement components C1q, C3 and C4 and the function of the classical complement pathway were normal in metastatic melanoma patients. Anti-alphaGal antibody titres were as variable in metastatic melanoma patients as in healthy controls, and the lysis of alphaGal-expressing cells correlated with anti-alphaGal immunoglobulin M titre (P < 0.0001). Anti-alphaGal antibody titres, complement levels and overall cytolytic function in the serum of patients with metastatic melanoma were indistinguishable from those of healthy controls. There is thus nothing intrinsic to the disease that will limit anti-alphaGal-based therapeutic strategies for enhanced antigen presentation or induced cell lysis, including the mimicry of hyperacute rejection.

Inactivation of murine leukaemia virus by exposure to visible light.

Prolonged storage of murine leukaemia virus in ambient light leads to a loss of infectivity. Particle integrity and envelope incorporation are unaffected; rather, the defect is functional and intrinsic to the viral core. Light in the violet part of the visible spectrum (wavelength 420-430 nm) is responsible for virus inactivation. Reduced reverse transcriptase-dependent cDNA generation post-entry accounts for the loss in infectivity and is likely due to a polymerase processivity defect. The virion-associated reverse transcription complex is thus photolabile. The phenomenon could be important in certain experimental situations, notably at elevated temperatures or when exposure to light is extensive. Additionally, our study suggests that the reverse transcription complex is a suitable target for an anti-retroviral strategy; identification of the nature of the lesion and the mechanism of its induction may inform the design of novel inhibitors.

Mice are unsuitable for modelling ABO discordance despite strain-specific A cross-reactive natural IgM.

ABO blood group antigens are immunodominant cell surface oligosaccharides. The function of the ABO system is clinically important in blood transfusion and solid organ transplantation but there is no small animal model of ABO discordance. The present study demonstrated A glycoconjugate-reactive IgM in the serum of CBA/Ca mice by enzyme-linked immunosorbent assay but showed with sugar blocking that the specificity of this IgM was different from that of human anti-A IgM. Furthermore, immunisation of CBA/Ca mice with the A antigen did not increase reactive IgM titre. In contrast, knock-out mice for the related carbohydrate antigen galactose(alpha1,3)galactose mounted a serum IgM response when immunised with the non-self galactose(alpha1,3)galactose antigen, which was shown to be T cell-dependent using a nude/knock-out animal. Reverse transcription-polymerase chain reaction identified transcripts for the enzyme likely to be responsible for the synthesis of the A antigen in organs from CBA/Ca mice although the A antigen was not detected in the same organs by immunohistochemistry. We conclude that CBA/Ca mice possess natural serum IgM with different characteristics to human anti-A IgM and that CBA/Ca mice may also express the A antigen. As a result, these mice are not suitable for use as a small animal model of ABO discordance.

In vivo efficacy of HSV-TK transcriptionally targeted to the tumour vasculature is augmented by combination with cytotoxic chemotherapy.

BACKGROUND: Retroviral vectors are suitable for targeting endothelial cells in the tumour neovasculature because of their intrinsic selectivity for proliferating cells. Previously, we inserted regulatory elements of the endothelial-specific prepro-endothelin-1 (ppET1) promoter in retroviral vectors to generate high-titre, replication-defective recombinant retroviruses that restricted gene expression to the vascular compartment of tumours. METHODS: A retroviral vector was generated in which expression of herpes simplex virus thymidine kinase (HSV-TK) was transcriptionally restricted to endothelial cells, under the control of a hybrid ppET-1 LTR. Xenograft tumour models were used to determine the efficacy of targeting HSV-TK to the tumour vasculature. Subsequently, vascular-targeted gene therapy was combined with chemotherapeutic agents. RESULTS: Breast or colorectal xenograft tumour growth was reduced and survival was increased in response to ganciclovir treatment. Treatment resulted in widespread vascular disruption and tumour cell apoptosis. In colorectal tumours, combination with irinotecan, a cytotoxic drug used to treat colorectal cancer, significantly increased survival compared to drug alone. No beneficial effect on survival was observed when combined with cisplatin, a cytotoxic drug not in clinical use for this tumour type. On the basis of their relative efficacies in vitro against tumour and endothelial cells, co-operativity with irinotecan likely derives from additionally targeting the peripheral tumour cells that survive the anti-vascular treatment. CONCLUSIONS: We show that the ppET1-targeted vector is efficacious for therapeutic gene expression in vivo, validating a strategy targeted to tumour vasculature, and demonstrate that vascular targeting combined with appropriate chemotherapy is more effective than either therapy alone.

Rescue of retroviral envelope fusion deficiencies by cationic liposomes.

BACKGROUND: Retroviral particles that are inappropriately enveloped can transduce target cells if pre-associated with cationic liposomes. This study optimises and addresses the mechanism of liposome-enhanced gene delivery, and explores the potential for such agents to compensate for fusion deficiency associated with chimaeric envelope proteins. METHODS: Particles bearing wild-type, chimaeric or no envelope proteins were complexed with DOTAP or DC-Chol/DOPE cationic liposomes and added to target cells for various times. Particle binding was determined by detection of cell-associated capsid protein and infectivity was measured histochemically. RESULTS: Stable association of cationic liposomes with retrovirus particles significantly enhanced their binding rate to target cells in proportion to the increase of transduction kinetics for infectious virus. Binding of virus was equivalent with or without envelope protein and/or virus receptor, indicating that a non-specific interaction precedes receptor recognition. Non-infectious combinations were rescued by the intrinsic fusogenicity of the cationic liposomes, which enabled entry of the viral core, but left subsequent events unaltered. The optimised transduction rate with non-enveloped particles and DOTAP approached that of amphotropic-enveloped virus in some cases, although the effect was target-cell-dependent. DC-Chol/DOPE was less potent at direct fusion but was able to enhance 600-fold the receptor-dependent action of chimaeric envelopes that were deficient in fusion by virtue of the addition of targeting domains. CONCLUSIONS: These data have implications for the development of retroviral vector targeting strategies from the perspectives of the specificity of target cell interaction and compensating for chimaeric envelope fusion deficiency.