Majid Mehtali

Gene therapy with recombinant adenovirus vectors: evaluation of the host immune response

E1, E3-deleted, replication-deficient recombinant adenoviruses are widely studied as vectors for their capacity to transfer therapeutic genes in vivo. They can infect a wide variety of dividing and quiescent cells from different organs and possess a large packaging capacity. One of the major limitations in the use of these vectors for gene therapy is the transient expression of the transgene in vivo and the poor transduction efficiency when re-administered. Despite the deletion of the viral E1 region, low level of early and late viral genes are expressed in vivo. Thus, viral antigens plus those derived from transgene expression in transduced cells contribute to cellular immune responses leading to the destruction of these cells. Production of anti-adenovirus antibodies, the cellular immune response as well as the early non-specific clearance of the vectors, constitute barriers to successful gene therapy. New vectors have been derived with additional deletions in the E2a or the E4 regions. Such second generation vectors were evaluated in vivo. These studies have revealed the complexity of the immune mechanisms elicited by these vectors and the importance of several parameters in these evaluations (i.e. mouse strains, nature of the transgene, route of administration...). In order to inhibit the production of neutralizing antibodies to adenovirus that prevent from further readministration of the vectors, immunosuppressive strategies were undertaken. Treatment regimens with immunosuppressive drugs (cyclophosphamide, FK506) or with monoclonal antibodies that block either the T cell receptor or costimulation pathways allow prolonged transgene expression and/or readministration of adenoviral vectors. In addition, transduction efficiencies may be increased by transiently inhibiting non-specific immune mechanisms that lead to the dramatic early clearance of the vectors. Taken together, these strategies may improve further gene therapy protocols by decreasing the host immune response to adenoviral vectors.

Immune Response to Recombinant Adenovirus in Humans: Capsid Components from Viral Input Are Targets for Vector-Specific Cytotoxic T Lymphocytes

ABSTRACT We previously demonstrated that a single injection of 109 PFU of recombinant adenovirus into patients induces strong vector-specific immune responses (H. Gahéry-Ségard, V. Molinier-Frenkel, C. Le Boulaire, P. Saulnier, P. Opolon, R. Lengagne, E. Gautier, A. Le Cesne, L. Zitvogel, A. Venet, C. Schatz, M. Courtney, T. Le Chevalier, T. Tursz, J.-G. Guillet, and F. Farace, J. Clin. Investig. 100:2218–2226, 1997). In the present study we analyzed the mechanism of vector recognition by cytotoxic T lymphocytes (CTL). CD8+ CTL lines were derived from two patients and maintained in long-term cultures. Target cell infections with E1-deleted and E1-plus E2-deleted adenoviruses, as well as transcription-blocking experiments with actinomycin D, revealed that host T-cell recognition did not require viral gene transcription. Target cells treated with brefeldin A were not lysed, indicating that viral input protein-derived peptides are associated with HLA class I molecules. Using recombinant capsid component-loaded targets, we observed that the three major proteins could be recognized. These results raise the question of the use of multideleted adenoviruses for gene therapy in the quest to diminish antivector CTL responses.

Porcine adenovirus-3 as a helper-dependent expression vector

Porcine adenovirus has been proposed as a potential vector for generating novel and effective vaccines for pigs. As a prerequisite for the generation of helper-dependent porcine adenovirus-3 (PAV-3) vectors, two E1-complementing porcine cell lines expressing E1 proteins of human adenovirus-5 (HAV-5) were made. These cell lines could be efficiently transfected with DNA and allowed the rescue and propagation of a PAV-3 recombinant, PAV201, containing a 0.597 kb E3 deletion and a 0.803 kb E1A deletion. Our data demonstrate that E1A proteins of HAV-5 have the capacity to transform foetal porcine retina cells and complement for the E1A proteins of PAV-3. The green fluorescent protein (GFP) gene placed under the control of a cytomegalovirus immediate early promoter was inserted into the E1A region of the PAV201 genome. Using these cell lines, a helper-dependent PAV-3 recombinant expressing GFP, PAV202, was constructed and characterized. The wild-type PAV-3 and the recombinant PAV202 expressing GFP were used to determine the ability of the virus to enter and replicate in cells of human and animal origin under cell culture conditions. Our results suggest that PAV-3 enters but does not replicate in dog, sheep, bovine and human cells.

Radioisotopic Imaging Allows Optimization of Adenovirus Lung Deposition for Cystic Fibrosis Gene Therapy

Cystic fibrosis is a common, heriditary disease resulting from mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Airway transfer of the CFTR gene is a potential strategy to treat or prevent the lung pathology that is the main cause of morbidity and mortality. Among the vectors used for gene therapy, adenoviruses have shown their ability to transfer the CFTR gene to respiratory epithelial cells, using either instillation or nebulization. Our objective was to characterize the lung deposition of aerosolized adenovirus by quantitative radioisotopic imaging, the only noninvasive technique allowing in vivo quantitation of inhaled drugs. We first labeled an adenovirus expressing human CFTR with the gamma-emitting radioisotope, technetium 99m (99mTc), and determined the best labeling conditions to allow preservation of virus bioactivity. We then administered the radioaerosol to baboons, determined lung regional deposition of 99mTc-labeled adenovirus, and compared the expression of CFTR transcripts 3 and 21 days after inhalation. The expression of vector-encoded mRNA ranged from 4 to 22% with respect to the endogenous CFTR mRNA depending on the lung segments. Moreover, we have developed a model using 99mTc-DTPA (diethylenetriamine pentaacetic acid), which can be used, as an alternative to adenovirus, to determine the profile of lung deposition of the vector. This study demonstrates that scintigraphy is a useful technique to achieve optimization of gene administration to the airways.

In Vivo Hepatocyte Retrovirus-Mediated Gene Transfer through the Rat Biliary Tract

Delivering retroviruses targeted to hepatocytes in vivo involves the injection of retroviruses directly into the blood stream of the portal vein. The aim of this work was to delineate the conditions for delivering retroviruses in vivo by perfusing in situ the bile duct of the regenerating rat liver, and to study the hepatocyte transgene expression. At 24 hr after partial hepatectomy, during the S phase of the cell cycle, regenerating livers were perfused for 2.8+/-0.5 hr through the bile duct with 36.2+/-6.8 ml (0.3+/-01 ml/min) of fresh culture supernatant containing amphotropic recombinant retroviruses encoding the beta-galactosidase gene. The virus total titer was 1.5 x 10(8) ffu (group I) or 6.5 x 10(8) ffu (groups II and III). The hepatic artery blood flow was either maintained (groups I and II) or interrupted (group III) during bile duct perfusion. Liver biopsies taken 7 days later showed that 31.4+/-24.2% (group I), 58.7+/-23.6% (group II), and 45.1+/-21.4% (group III) of hepatocytes expressed beta-galactosidase activity, predominantly in the periportal and mediolobular zones. This study demonstrates that hepatocytes of regenerating rat livers that have entered the S phase of the cell cycle as a result of partial hepatectomy can be transduced in vivo by retroviral vectors delivered in situ by bile duct perfusion. Furthermore, the number of transduced hepatocytes closely correlated with the viral total titer and was diminished by hepatic artery blood flow occlusion during perfusion.

Experimental gene therapy : the transfer of Tat-inducible interferon genes protects human cells against HIV-1 challenge in vitro and in vivo in severe combined immunodeficient mice

Objectives: To evaluate in vitro and in vivo a strategy for gene therapy for AIDS based on the transfer of interferon (IFN)‐&agr;, &bgr; and ‐&ggr; genes to human cells. Design: Human U937 promonocytic cells were stably transfected with Tat‐inducible IFN expression vectors conferring an antiviral state against infection with HIV. Methods: Transfected cells were either infected by HIV‐1 in vitro or transplanted into severe combined immunodeficient (SCID) mice for an HIV challenge in vivo. Results: U937 cell lines stably carrying IFN transgenes under the positive control of the HIV‐1 Tat protein were highly resistant to HIV‐1 replication in vitro. This antiviral resistance was associated with a strong induction of IFN synthesis immediately following the viral infection. HIV‐1 proteins were found to be specifically trapped within the genetically modified cells. In contrast, all IFN‐U937 cells permitted full HIV‐2 replication. Transfected cells injected into SCID mice and challenged against HIV‐1 were strongly resistant to infection when cells were transduced with IFN‐&agr; or IFN‐&bgr; genes. However, IFN‐&ggr;‐transfected cells permitted HIV‐1 infection in vivo despite the induction of a high level of IFN‐&ggr; secretion. The quantity of proviral DNA was 105‐fold lower in IFN‐&agr;‐ or IFN‐&bgr;‐transfected U937 cells collected from these SCID mice than that in non‐transfected cells. Conclusions: Our results substantiated the validity of a strategy, based on the transfer of HIV‐1‐inducible IFN‐&agr; or IFN‐&bgr; genes, to confer antiviral resistance to human cells.

DELETION OF THE FIBER GENE INDUCES THE STORAGE OF HEXON AND PENTON BASE PROTEINS IN PML/SP100-CONTAINING INCLUSIONS DURING ADENOVIRUS INFECTION

The present study has documented changes in the in situ distribution of viral DNA and capsid proteins in 293 cells infected with fiber gene‐deleted adenoviruses. It shows that infection results in the intense production of progeny viruses which appear morphologically intact although they are devoid of fiber‐coding sequence in their genome and hence of fiber protein in their capsid. The data confirm, therefore, that fiber protein is not essential for the assembly of progeny viruses. The main contribution of our observations concerns specific intranuclear structures induced by infection with either wild‐type or fiber gene‐deleted viruses. These clear amorphous inclusions contain two cellular proteins, PML and Sp100, which in non‐infected cells co‐localize to a special type of nuclear bodies. PML and Sp100 nuclear bodies appear to directly modulate or to be altered in a wide variety of situations including viral infections, cell death and transformation. In cells infected with fiber gene‐deleted viruses, the clear amorphous inclusions now accumulate non‐used hexon and penton base proteins, whereas the absence of fiber protein prevents the assembly of capsid proteins in crystallin arrays. Taken together, the data suggest that the clear amorphous inclusions may correspond to storage sites of structural and regulatory proteins. Consequently, these virus‐induced structures may promote the productive cycle of adenoviruses by regulating the amount of over‐produced viral proteins and the shutoff of the host cell metabolism.

LOCALIZATION OF VIRAL PROTEIN X IN SIMIAN IMMUNODEFICIENCY VIRUS MACAQUE STRAIN AND ANALYSIS OF ITS PACKAGING REQUIREMENTS

Simian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) encode the accessory viral protein X (Vpx) known to be incorporated into virions in amounts comparable to those of the Gag proteins. The localization of Vpx within SIVmac-infected HUT-78 cells and SIVmac virions was studied by immunoelectron microscopy. Vpx appeared to be associated with extracellular virions as well as budding viral particles at the surface of infected cells. Immunolabelling of purified viral cores suggested that Vpx was a component of the amorphous material surrounding the core structure. Furthermore, a detergent insoluble fraction containing SIV core proteins was devoid of Vpx. To investigate the protein requirement for packaging of Vpx, BHK-21 cells were co-infected with vaccinia virus recombinants encoding Vpx and other SIV proteins able to assemble into virus-like particles. Analysis by immunoprecipitation of the extracellular particulate material as well as immunoelectron microscopy demonstrated that co-expression of Vpx with the Pr56gag polyprotein was sufficient for the formation of pseudo-virions containing Vpx. Virus-like particles that appeared upon expression of p16gag did not contain Vpx. The results suggest that Vpx is packaged into viral particles through its binding to the Gag polyprotein. The precise positioning of Vpx within the space separating the viral envelope from the core structure is postulated to result from the reorganization of viral proteins that occurs upon Gag polyprotein cleavage and budding.

A preclinical model of hepatocyte gene transfer: the in vivo, in situ perfused rat liver

Delivering retroviruses targeted to hepatocytes in vivo involves the injection of retroviruses directly into the portal vein. The aim of this work was to establish a clinically relevant system for retrovirus-mediated gene transfer in a new model of in vivo, in situ perfused rat liver and to study the transgene expression. At 24 h after partial hepatectomy, the liver was completely excluded from the splanchnic circulation using an extracorporeal shunt. Two independent normothermal, oxygenated perfusion systems were used. First, liver perfusion was carried out with a recirculating system (1 h). Culture supernatant containing retroviruses (1.5 × 108 ffu/ml, β-galactosidase gene) was used as perfusate. Then the liver perfusion was maintained for more 30 min in a single liver passage system using culture medium without retroviruses as perfusate. High hepatocyte transduction rates (up to 34.4%) were obtained. PCR analysis showed no provirus in extrahepatic organs. Viral titrations performed simultaneously (inflow and outflow liver lines) showed that after 1 h of perfusion (up to 30 successive liver passages) retroviruses were still detected in the liver outflow perfusate (up to 2.0 × 107 ffu/ml). Washing the liver for 30 min dramatically decreased the leakage of retroviruses in the outflow. In order to be of clinical use, the injection of retroviruses targeted to hepatocytes in vivo should be done while the liver is completely excluded from the splanchnic circulation to avoid any extrahepatic retrovirus diffusion.