Sendai Virus Vectors without All of the Envelop-Related Genes: Successful Recovery and Characterization of the Three Genes (Matrix, Fusion and Hemagglutinin-Neuraminidase)-Deleted Vectors

RNA VIRUS VECTORS III Administration of the peptides did not elevate expression levels over levels measured in these control groups. This result indicates either that T cell responses need to be further reduced or that immune mechanisms other than T cells play a major role in eliminating SeV. *both authors contributed equally to this work

722. SeV RNA Replicon Vector: An Efficient Cytoplasmic Expression System Derived from Sendai Virus Self-Replicating Ribonucleoprotein Complexes Akihiro Iida,1 Mamoru Hasegawa.1 DNAVEC Research Inc., Tsukuba, Ibaraki, Japan.

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721. Proper Maturation of CFTR Protein Expressed by SeV Vector Was Proved by Using a GFP-CFTR Fusion Protein Hiroshi Ban,1 Makoto Inoue,1 Uta Griesenbach,2,4 Jun You,1 Steve C. Hyde,3,4 Eric W. F. W. Alton,2,4 Akihiro Iida,1 Mamoru Hasegawa.1 1 DNAVEC Research Inc., Tsukuba, Ibaraki, Japan; 2Gene Therapy, NHLI, Imperial College, London, United Kingdom; 3 Clinically Laboratory Sciences, NDCLS, University of Oxford, Oxford, United Kingdom; 4UK Cystic Fibrosis Gene Therapy Consortium. Sendai virus (SeV) vector has been shown to transduce the airway epithelial cells very efficiently. This property makes the vector promising for cystic fibrosis (CF) gene therapy. Previously, we generated a series of SeV vectors carrying the cystic fibrosis transmembrane conductance regulator (CFTR) gene and showed that SeV can mediate CFTR gene transfer both in vitro and in vivo. CFTR activity in such experiments was confirmed in C127 cells in vitro using a radioactive iodide efflux assay and a whole cell perforated patch-clamp assay, and in CF knockout mice in vivo. These results indicated that SeV vector was a good candidate vector for CF gene therapy. However, there was not clear correlation between the expression levels of CFTR estimated from the design of a series of the vectors and observed activities of them. Additionally, cytotoxicity was observed in cells transduced by some types of SeV vectors probably or largely because of the overexpression of CFTR. To clarify these matters, biochemical analysis for CFTR protein itself is indispensable because CFTR is known to be highly glycosylated and glycosylation is responsible for mature CFTR function. However, we could not detect the CFTR by Western blotting using anti-CFTR antibody in SeV vector-transduced cells. Thus, we constructed a recombinant SeV vector designated SeV(HNL)GFPCFTR/∆F in which the N-terminus of CFTR was fused with an useful tag, GFP gene. The fusion gene was introduced into the junction between the hemagglutinin-neuraminidase (HN) and large protein (L) genes on the F gene-deleted SeV (SeV/∆F) genome. It has previously been shown that GFP fusion to the N-terminus of CFTR did not affect processing, localization and function of CFTR (Bryan D. et al. 1998, J Biol Chem 273, 21759-68). We characterized the GFP-CFTR in the cells transduced with SeV(HNL)-GFPCFTR/ ∆F. Western blotting using an anti-GFP antibody detected two proteins at 190 kDa and 170 kDa. These correspond in size to fully glycosylated mature CFTR and to the core-glycosylated immature protein, respectively. The mature and immature CFTR are known to be distinguishable by their different sensitivity to glycosidases. As expected, the 190 kDa protein was sensitive only to PNGaseF glycosidase and 170 kDa protein was to both PNGaseF and EndoH glycosidase. These results demonstrate that glycosylation of GFPCFTR fusion protein derived from SeV(HNL)-GFPCFTR/∆F was quite similar to endogenous CFTR protein. The subcellular localization of GFP-CFTR in MDCK cells infected with SeV(HNL)GFPCFTR/∆F was further analyzed by confocal laser microscopy. The GFP-CFTR was found to predominantly localize to the apical membrane. Thus, the proper maturation of CFTR protein in SeV vector-transduced cells was proved, indicating again that SeV is useful for CF gene therapy. Additionally, the generated SeV(HNL)GFPCFTR/∆F would be an useful system to analyze dynamics of CFTR protein including the tracking both in vitro and in vivo. The quantitative and functional analyses are now under investigation. Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright © The American Society of Gene Therapy

Sendai virus (SeV) is an enveloped virus with a nonsegmented negative strand RNA belonging to Paramyxoviridae family. We have previously shown that recombinant SeV as well as its modified nontransmissible variants can efficiently transfer and express foreign genes for a broad range of mammalian cells and tissues in vitro and in vivo and are suitable for use in gene therapy. SeV has two envelope glycoproteins HN and F, which mediate the virus attachment to and penetration into target cells, respectively. These envelope glycoproteins are not only important factors for viral tropism but also major determinants of immunogenicity in infected animals. Here we report the development of a novel SeV RNA replicon vector system that takes advantage of SeV self-replicating ribonucleoprotein complexes (RNP). In the vector system, RNP itself is non-infectious and can be infectious by the addition of lipofection reagents. In this system, nuclear entry of SeV RNA genome which forms an RNP complex with SeV NP, P and L proteins is not required and high expression of transgene is expected in transduced cells by selfreplication of RNP in the cytoplasm. In addition, no effect of transduction of transgene in the presence of neutral antibody against SeV is expected. As the source of RNP, non-infectious virus-like particles (VLP) were recovered by infecting F-defective SeV vector into LLC-MK2 cells and harvesting VLP from the supernatants of the infected cells. To qualitatively and quantitatively compare this new vector system with conventional SeV vector or plasmid DNA, we introduced β-galactosidase, luciferase or enhanced green fluorescent protein (EGFP) reporter gene into the F-defective SeV genome, and recovered VLP from cloned cDNAs. The SeV RNA replicon vector mixed with lipofection reagents efficiently transferred foreign genes to several mammalian cell lines in vitro, and dosedependently expressed luciferase or EGFP genes in tranduced cells. The expression level of SeV RNA replicon vector was comparable to that of SeV F-defective vector and remarkably higher than plasmid DNA-liposome complexes. Transduction of a foreign gene by SeV RNA replicon vector was not inhibited by the addition of an antiSeV-HN antibody, although transduction by conventional SeV vector was completely inhibited. Therefore, the SeV RNA replicon vector system is useful for cytoplasmic expression of foreign genes in vitro and may be of help to its application to human gene therapy such as repeated administration and targeting of the vector.

723. Sendai Virus Vectors without All of the Envelop-Related Genes: Successful Recovery and Characterization of the Three Genes (Matrix, Fusion and Hemagglutinin-Neuraminidase)Deleted Vectors Makoto Inoue,1 Mariko Yoshizaki,1 Yumiko Tokusumi,1 Takashi Hironaka,1 Hiroshi Ban,1 Yoshiyuki Nagai,2 Akihiro Iida,1 Mamoru Hasegawa.1 1 DNAVEC Research Inc., Tsukuba, Ibaraki, Japan; 2Toyama Institute of Health, Toyama, Japan. Sendai virus (SeV) vector is a new class of vector bearing a new concept of “cytoplasmic RNA vector”. This vector based on SeV belonging to the genus Respirovirus of the family Paramyxoviridae, infects and replicates in most mammalian cells, and directs highlevel transgene expression. Its replication is independent of nuclear functions and does not have a DNA phase during its life cycle so that the transformation of cells by integration of vector materials S275

RNA VIRUS VECTORS III into the cellular genome is not a concern. These properties make SeV vectors very promising for application to gene therapy and vaccination via the expression of therapeutic genes and antigens. We have previously succeeded in the recovery of fusion (F) gene-deleted SeV vector (SeV/∆F), matrix (M) gene-deleted SeV vector (SeV/ ∆M), both M and F genes-deleted SeV vector (SeV/∆M∆F) and other types of the vectors in high titers. F gene-deletion made SeV vector non-transmissible, and M gene-deletion worked well on the vector to become incapable of formation of the particles from infected cells. All of the above SeV vectors maintain efficient infectivity in vitro and in vivo as wild type SeV does. The hemagglutinin-neuraminidase (HN) protein, which mediates the attachment of SeV, is known to be one of the major targets for host immune responsive machineries on SeV infection such as natural killer cell (NK), cytotoxic T lymphocyte (CTL) and neutralizing antibodies. The HN gene-deletion from the genome would be one of the important ways to reduce the immune response against SeV vector. Therefore, the HN gene was further deleted from the genome of SeV/∆M∆F, and thus SeV/∆M∆F∆HN was generated. The SeV/ ∆M∆F∆HN possesses only nucleoprotein (NP), phosphoprotein (P) and large protein (L)-genes on its genome. All of them are essential for efficient expression of SeV vector by consisting ribonucleoprotein (RNP) complex. The new vector was successfully recovered and propagated in a new packaging cell line expressing M, F and HN proteins by using a Cre/loxP inducible system. The titer of SeV/ ∆M∆F∆HN carrying the enhanced green fluorescent protein (GFP) was and above 1x107 cell infectious units (CIU)/ml. This vector showed efficient infectivity and gene expression in various types of cell lines and primary cells in vitro. When this vector was administered into the auricle of the ear of mice (5x106 CIU), a visible transduction was confirmed as in the cases of wild type SeV vector in vivo. The important investigation for immune reaction of this vector is in progress. The SeV/∆M∆F∆HN vector is the most advanced type of cytoplasmic RNA virus vector and the like Sendai virus vector.

724. Engineering of Vesicular Stomatitis VirusG Protein (VSV-G) To Retarget Recombinant Infectious Retroviral Particles Hanna S. Dreja,1 Marc Piechaczyk.1 1 Institut de Génétique Moléculaire/UMR 5535, IFR24, CNRS, Montpellier, France. Introduction Recombinant retroviruses, including lentiviruses, are the most widely used vectors for both in vitro and in vivo stable gene transfer. However, this technology suffers from a number of drawbacks, which limits its application. One of the main restrictions is the inability to deliver transgenes specifically into cells of interest, which would increase efficacy, safety and reduce the costs of gene therapy treatments. One way of achieving this is to modify the host range of retroviral vectors, where the challenge lies in constructing chimaeric proteins, which can be efficiently incorporated and retained in viral particles, recognise targeted markers at the surface of cells of interest, show high fusion activity and be used to modify the tropism of many different vector types. We propose that VSV-G could be the ideal prototype for such chimaeric viral proteins. VSV-G is one of the most fusiogenic viral proteins identified and can be incorporated into many enveloped viruses, which can then efficiently infect a wide array of cells. Importantly, VSV-G is physically stable, which limits loss of viral infectivity over time and permits concentration of virus stocks by centrifugation. Here, we build a model system for VSV-G modification, where a cell-directing single-chain antibody (scFv) is linked to the VSV-G backbone. S276

Methods Chimaeric VSV-G molecules were engineered by grafting cDNAs of scFv (anti-MHC class I (MHC) or anti-hen egg lysosyme (HEL)) in the N-terminal of the VSV-G sequence. Expression of VSV/scFvs in transiently transfected 293T cells was assessed by Western Blot experiments. Intracellular localisation was analysed following immunofluorescent labelling assays. Fusion potentials of the proteins were determined using transfected HeLa cells exposed to a low pH fusion medium. Incorporation into murine leukaemia- or lentiviral vectors was examined following concentration of viral supernatants from transfected TelCeb6- or 293T cells, respectively. VSV/scFvpseudotyped lentiviruses were titered on human target cells. Results & Conclusion We could confirm that the VSV/scFvs were expressed in transiently transfected cells, and distributed in the proximity of the nuclei, in the cytoplasm and plasma membrane, similar to that of the parental molecule. Also, incorporation into retroviral particles was achieved. However, the fusion capabilities of the modified proteins were dramatically reduced and the lentiviral infection titers were considerably decreased. Yet, interestingly, initial findings suggest that we have a selective advantage for infections of human cells with VSV-MHC pseudotyped lentivirus, as compared to control chimaera. The inherently low titers may be improved by inclusion of a cleavable site between the grafted peptide and the viral component. Also, infection experiments are currently being carried out on non-human cells. To our knowledge, this is the first attempt to change the infection profile of VSV-G pseudotyped vectors.

725. Engineering Enhanced Retroviral Vectors by Random Insertion of Functional Peptides Julie H. Yu,1 David V. Schaffer.1 Chemical Engineering, University of California, Berkeley, Berkeley, CA.

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Several obstacles still need to be overcome before retroviral vectors can be used in a wide clinical application. Engineering of these viruses by inserting novel protein or peptide sequences into several viral proteins could readily enhance their properties and features for gene delivery. However, it is difficult to rationally determine where within a protein to insert a new sequence without disrupting the core ability of the virus to transduce cells. We have developed a system to modify viral genomes through the random insertion of small peptides that may confer novel function to the viral vector. This high throughput method generates a library of proteins that have a specifically designed peptide or protein sequence incorporated at as many or all possible points in the sequence of the original protein. We can then use a directed evolution approach and screen these libraries to identify tolerable insertion sites that may yield a desired function. We have used this method to generate insertional libraries of two important genes: the Moloney murine leukemia virus (MoMLV) gag-pol gene, which encodes for most of the structural and functional proteins of the virus, and the gene that encodes for the vesicular stomatitis virus G protein (VSV-G), an envelope protein commonly used to pseudotype simple retroviral and lentiviral vectors. We are using these libraries to explore the addition of novel features to MoMLV for gene delivery applications. These features address some of the major limitations of retroviral vectors including delivery to non-dividing cells, tissue specific targeting, and purification. Identification of mutants with enhanced function will allow us to generate a more effective and efficient retroviral vector. Furthermore, the detailed genetic footprint provided by the comprehensive coverage of insertion sites will give us additional knowledge of the function of these specific genes and their associated proteins. Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright © The American Society of Gene Therapy