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Volume 6 - 2001


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1-24

Mammalian genome organization and its implications for the development of gene therapy vectors

Author(s): Dr. Daniele Zink,

Abstract: The transcription of mammalian genes and transgenes integrated into mammalian genomes is regulated at three different levels: the molecular level (comprising the interaction of transcription factors with specific DNA elements), the level of chromatin structure, and the level of nuclear architecture. Transcriptional regulation of integrating gene therapy vectors is only well investigated at the molecular level, few data exist regarding the involvement of chromatin structure, and virtually nothing is known about the involvement of nuclear chromosome- and genome architecture. Therefore, it is not surprising that the expressional behavior of gene therapy vectors after integration is often unpredictable and difficult to improve. This review will outline, after giving an overview of recent results and concepts concerning mammalian genome architecture, how this level of organization might be involved in the transcriptional regulation of integrating vectors. First results will be presented and the implications for future vector development will be discussed.

Keywords: Nuclear architecture, genome architecture, chromosome structure, interphase chromosome territory, nuclear positioning, nuclear localization, chromatin structure, genome dynamics, integration site, integrating gene therapy vector



25-31

Novel developments for applications of alphavirus vectors in gene therapy

Author(s): Dr. Kenneth Lundstrom,

Abstract: As a prerequisite for gene therapy applications, alphavirus-mediated delivery of reporter genes to different brain regions in rodents has resulted in local, high-level expression of a transient nature. Infection of human prostate tumor cell lines with recombinant Semliki Forest virus (SFV)-LacZ particles demonstrated a strong induction of apoptosis that led to premature cell death. Injection of self-replicative SFV-LacZ RNA showed in prophylactic and therapeutic effects in animals. Furthermore, injection of SFV vectors expressing interleukin-12 resulted in tumor regression in a mouse B16 melanoma tumor model. Similarly, injection of SFV vectors expressing GFP, β- galactosidase or even empty SFV vectors led to p53-independent induction of apoptosis in nude mice with implanted human lung carcinomas. Repeated injections showed improved anti-tumor responses without any visible immune reaction against injected SFV particles. The envelope structure of alphaviruses has been modified to allow cell/tissue specific targeting. Moreover, SFV vectors have been used for the production of retrovirus-like particles. Extensive development on alphavirus vectors has resulted in novel non-cytopathogenic and replication-persistent forms. Overall, alphavirus vectors can be considered highly attractive for future gene therapy applications.

Keywords: Semliki Forest virus, gene therapy, vaccine, LacZ, cell targeting, gene expression



33-46

We have recently introduced an episomally replicating vector the function of which depends on the combination of SV40 origin of replication with a human scaffold/matrix attached region (S/MAR). The episomal status of this vector is maintained in several cell lines for an extended period of time in the absence of a virally encoded protein and in the absence of selection conditions. In this article we start to identify the elements required for recruiting this type of episome to the endogenous cellular replication machinery and we discuss aspects of replication-transcription coupling. We try to establish a catalogue of parameters which should be considered for the design of functional episomes.

Author(s): Dr. Jürgen Bode,

Abstract: We have recently introduced an episomally replicating vector the function of which depends on the combination of SV40 origin of replication with a human scaffold/matrix attached region (S/MAR). The episomal status of this vector is maintained in several cell lines for an extended period of time in the absence of a virally encoded protein and in the absence of selection conditions. In this article we start to identify the elements required for recruiting this type of episome to the endogenous cellular replication machinery and we discuss aspects of replication-transcription coupling. We try to establish a catalogue of parameters which should be considered for the design of functional episomes.

Keywords: Hitchhiking principle, gene therapy, biotechnology, replication origins, mammalian viruses, Maintenance elements, Replication–transcription coupling, nonviral episomes



47-55

Mammalian genome organization and its implications for the development of gene therapy vectors

Author(s): Dr. Yosef Gruenbaum,

Abstract: The nuclear pore complex (NPC) is the site for macromolecular traffic between the nucleus and cytoplasm. NPCs are composed of 30-40 distinct proteins (termed nucleoporins) in yeast, and an estimated 50 distinct nucleoporins in vertebrates. Most nucleoporins are soluble proteins. In contrast, the number of integral membrane nucleoporins is small and includes only four proteins in yeast (Pom152, Ndc1, Snl1 and Pom34), and two proteins in metazoans (gp210 and Pom121). We discuss the known membrane nucleoporins, and present for the first time the sequences of putative gp210 orthologs from Drosophila, C. elegans and Arabidopsis. Our results suggest that Gp210 is conserved among all multicellular eukaryotes, including plants, consistent with a fundamental role in NPC structure or biogenesis.

Keywords: Nucleocytoplasmic trafficking, nucleoporins, gene therapy vectors



57-67

Design and construction of oncoretroviral vectors expressing a packageable ribonuclease for use in HIV gene therapy

Author(s): Dr. Sadhna Joshi,

Abstract: A number of different strategies are being developed for inhibition of human immunodeficiency virus type-1 (HIV- 1) replication via gene therapy. In this study, a packageable ribonuclease, Gag-RNase T1, was constructed. The Gag domain from HIV-1 should allow copackaging into HIV-1 virions and the RNase T1 domain from Aspergillus oryzae should allow cleavage of HIV-1 virion RNA. In order to have regulator of expression of virion proteins (Rev)- dependent and Rev-independent production of Gag-RNase T1, the HIV-1 Rev responsive element (RRE) and the mason-pfizer monkey virus (MPMV) constitutive transport element (CTE) were cloned downstream to the gagt1 gene. Expression and enzymatic activity of the Gag-RNase T1 fusion protein was compared using the Moloney murine leukemia virus (MoMuLV)-based vector, MoTiN, and murine stem cell virus (MSCV)-based vector, MGIN. Very little amount of Gag-RNase T1 was present in the cell lysate and in the culture supernatant of cells co- transfected with the MoTiN-based vector. In contrast, the amount of Gag-RNase T1 present in the cell lysate and in the culture supernatant of cells co-transfected with the MGIN-based vector was ~20 fold better. HIV-based lentiviral vector particles produced from cells expressing Gag-RNase T1 or mutant Gag-RNase T1 were also analyzed. Gag-RNase T1 present in these samples was shown to be full-length (56 kDa) and was enzymatically active in vitro. However, the titer of these vector particles was not decreased. These results suggest that Gag-RNase T1 is only capable of homochimeric assembly and is excluded from vector particles containing the HIV-1 Gag and Gag-Pol proteins.

Keywords: HIV-1 Gag, Packageable nuclease, RNase T1, Moloney murine leukemia virus-based vector, murine stem cell virus-based vector.



69-77

Tat-RNase H and its use in HIV gene therapy

Author(s): Dr. Sadhna Joshi,

Abstract: A targeted RNase, Tat-RNase H, was designed and tested for its activity in vitro and inhibition of HIV-1 replication in vivo. The Tat-RNase H protein consists of the TAR (trans-activation response) element-binding domain of the HIV-1 Tat (trans-activator of transcription) and the RNase H domain of the HIV-1 reverse transcriptase (RT) (Melekhovets and Joshi, 1996). The Tat protein binds specifically to the TAR element present in all HIV-1 RNA molecules, whereas HIV-1 RNase H specifically degrades RNA within RNA/DNA hybrid in vivo (Skalka and Goff, 1993; Telesnitsky and Goff, 1997) and to a lesser degree within RNA/RNA hybrid in vitro (Ben-Artzi et al, 1992; Gotte et al, 1995). Thus, there are two anticipated modes of action of the Tat-RNase H protein. It could cleave HIV- 1 TAR RNA in RNA/RNA hybrid or in RNA/DNA hybrid. RNA cleavage in RNA/RNA hybrid was previously shown to be specific and to depend on interaction between the Tat domain of Tat-RNase H and the TAR element of HIV-1 RNA (Melekhovets and Joshi, 1996). We demonstrate here that the Tat-RNase H mediated cleavage of RNA in RNA/DNA hybrid is non-specific as both TAR and mutant TAR RNA/DNA hybrids could be efficiently cleaved. A retroviral vector expressing Tat-RNase H was then constructed to assess whether Tat-RNase H can inhibit HIV-1 replication. However, the Tat-RNase H protein failed to inhibit HIV-1 replication in transduced MT4 cells and in peripheral blood T lymphocytes (PBLs). The possible reasons why Tat-RNase H might have failed to inhibit HIV-1 replication in MT4 cells and PBLs are discussed.

Keywords: Gene therapy, HIV-1, retroviral vector, targeted RNase.



79-89

DNA Vaccination for the induction of immune responses against HIV-1 subtype C envelope gene in mice

Author(s): Dr. Pradeep Seth,

Abstract: human primate models is warranted. Gene Therapy and Molecular Biology Vol 6, page 79 Most human immunodeficiency virus (HIV) DNA vaccines currently being developed are based on clade B strains of HIV-1, which are found predominantly in North America and Europe. Since in India, subtype C is the predominant strain of HIV-1, it is imperative that a vaccine based on the local circulating subtype should be designed. Two DNA constructs encoding HIV-1 envelope glycoprotein (gp120) obtained from HIV-1 subtype C primary isolates were used for immunising mice. Mice immunised intramuscularly with these constructs produced low levels of antibodies against Gp120. However, these animals showed MHC class I restricted cytotoxic T lymphocyte (CTL) activity against homologous (subtype C) as well as heterologous (subtype B) peptide pulsed target cells thus demonstrating cross clade reactivity. In addition, in vitro lymphocyte proliferation and Th1 cytokine response to HIV antigen stimulus was seen with high levels of IFN-? and IL-2 but undetectable IL-4 and IL-5 production. These findings indicate that these constructs have possible value as potent vaccines. Their further characterization in non-

Keywords: HIV-1, subtype C, DNA vaccine, cross reactivity, cell mediated immunity, HIV vaccine



91-99

Sustained tissue -specific transgene expression from a vl30 retrotransposon-derived vector in vivo

Author(s): Dr. Clague P. Hodgson,

Abstract: Previous attempts to generate transgenic mice via retroviral transduction of pre-implantation embryos have usually not resulted in stable transgene expression. In these cases, inactivation of the retroviral LTR is associated with passage through the germ line. A subset of endogenous murine retrovirus-like retrotransposons (VL30’s) are constitutively expressed in virtually all tissues, with no deleterious effects to the health of the animal. We surmised that these VL30s might be useful as vectors for stable lineage-specific transgene expression. A mouse VL30-derived retro-vector engineered to express a reporter gene (LacZ) was used to generate transgenic mice via transduction of embryonic stem (ES) cells. A single copy of the vector was stably integrated into a unique site in the mouse genome. Sustained tissue specific expression was observed at both the mRNA and protein levels for several generations. Transgene expression was observed in distinct sub-populations of cells in both lung and spleen. In the lung, cells expressing the vector were identified as type II pneumocytes. These data illustrate for the first time that a VL30 LTR (NVL-3) is unique from its retroviral counterparts in that it can pass through the germ line repeatedly without undergoing transcriptional inactivation. Thus, VL30 vectors may be useful for both transgenesis and as alternatives to existing retroviral vectors for gene therapy.

Keywords: VL30 retrotransposon, transgenic, sustained expression



101-119

Cytokine gene transduced T cells in the treatment of allergic encephalomyelitis and airway hypersensitivity

Author(s): Dr. Gerald Hochwald,

Abstract: Summary TGF-β1 or IL-10 transduced myelin basic protein (MBP)-specific BALB/c cloned Th1 cells were injected into SJL x BALB/c F1 mice 11-15 days after immunization with proteolipid protein to induce EAE. TGF-β1/MBP T cells significantly ameliorated the EAE, while IL-10/MBP T cells were less effective. TGF-β1 transduced ovalbumin (OVA)-specific Th1 clones did not influence EAE, even when re-activated by OVA in vivo. However, TGF-β1/OVA T cells did protect against OVA-specific Th2-cell mediated airway hyper-reactivity induced by inhaled OVA. TGF- β1/KLH T cells did not prevent OVA-induced airway hyper-reactivity in mice sensitized and challenged with OVA alone, but did protect mice challenged with KLH + OVA. Thus, the antigen specificity of the Th1 cells allows site- specific delivery of therapeutic TGF-β1 to both Th1 and Th2 cell-mediated inflammatory infiltrates. EAE relapses, induced by bacterial superantigen or endotoxin within 2 weeks, but not >6 weeks, after transfer of TGF-β1 or IL- 10/MBP T cells, were reduced. Relapses induced 5 weeks after immunization with PLP could be prevented by simultaneously injected TGF-β1/MBP cells. Spinal cords taken 12-50 days after TGF-β1/MBP cells contained TGF- β1 cDNA. Spinal cords from the majority of mice receiving IL-10/MBP cells contained IL-10 cDNA up to 2 weeks, but not 50 days after cell transfer. Thus, TGF-β1-transduced T cells may be useful in the therapy of autoimmune and allergic inflammatory diseases, but in the EAE model, the same approach with IL-10-transduced T cells appears less effective.

Keywords: autoimmunity, cytokines, gene therapy, experimental allergic encephalomyelitis, Th1/Th2.



121-131

The adenine nucleotide translocator as a potential therapeutic target

Author(s): Dr. Dr. C. Brenner,

Abstract: Identification of new targets for development of apoptosis-modulating drugs has become possible from the unraveling of the basic apoptosis mechanisms. Thus, mitochondrial membrane permeabilization has been recently recognized as a central rate-limiting step of apoptosis and its study has led to the identification of the adenine nucleotide translocator (ANT) as a potential therapeutic target. Three arguments support this possibility. First, ANT is a bi-functional protein, an ADP/ATP translocator and a non-specific pore, which contributes to apoptosis via its capacity to form a lethal pore under the control of the Bax/Bcl-2 family members. Second, the pore-forming activity of ANT is directly modulated by agents as diverse as proteins, lipids, ions, pro-oxidants or chemotherapeutic agents. Third, loss of ANT function is involved in several human pathologies, such as cardiomyopathy and aging, while reduced ANT expression or ANT mutation may lead to renal cancer and ophtalmoplegia. Hypothetically, ANT may thus constitute a new target for therapeutic intervention on apoptosis.

Keywords: Bcl-2, drug design, gene therapy, liposome, mitochondrion, oncogene



133-141

Reticuloendotheliosis virus-derived vectors for human gene therapy

Author(s): Dr. Ralph Dornburg,

Abstract: Spleen necrosis virus (SNV) and reticuloendotheliosis virus strain-A (REV-A) belong to the family of avian reticuloendotheliosis viruses (REV). These amphotropic retroviruses infect a large variety of cells of avian and some mammalian species. SNV or REV-A with wild-type envelope does not infect human cells. However, they efficiently infect and integrate their genome into that of human cells when they are pseudotyped with the envelope protein of other mammalian retroviruses or the G protein of vesicular stomatitis virus (VSV). Moreover, SNV-derived retroviral vectors, which display single chain antibodies on the viral surface, enable cell-type-specific gene delivery into various human cells. In particular, the SNV cell-type-specific gene delivery vector system appears to be very well suited to transduce genes into cells of the human hematopoietic system. Moreover, my laboratory has developed genetically engineered SNV vectors, which are capable of infecting non-dividing cells such as quiescent human T-cells and primary monocyte-derived macrophages. Thus, REV-derived vectors appear to be very interesting candidates for the development of vectors for human gene therapy. The biology, genomic organization, and replication of these viruses have been reviewed in detail previously (Dornburg, 1995). Thus, this review focuses on the recent progress in the developments of REV-derived vectors for gene transfer into human cells.

Keywords: spleen necrosis virus, reticuloendotheliosis virus, retroviral vectors, helper cells, gene therapy



143-148

Design of methacrylate-based polyplexes for tumor targeting

Author(s): Dr. Gert Storm,

Abstract: A non-viral gene delivery vector has been developed in our laboratory based on the cationic polymer poly(2- (dimethylamino)ethyl methacrylate) (p(DMAEMA)). This contribution deals with the design of pDMAEMA-based polyplexes for tumor targeting. The first part is concerned with their use for the intraperitoneal therapy of ovarian cancer, the second part with their use for intravenous targeting of solid tumors. It is demonstrated that cell-specific gene delivery to in vitro cultured ovarian carcinoma cells can be obtained by coating p(DMAEMA)-based polyplexes with an anionic lipid layer bearing conjugated antibody fragments. As the lipid coat around the so-called lipopolyplexes (LPP) efficiently shields the positive charge of polyplexes, the predominant electrostatic interaction with cell membranes could be avoided. As LPP without antibody did not show transfection, it can be concluded that the presence of a targeting ligand is essential. In addition, the lipid coat around the LPP provided protection of the polyplexes against destabilization by polyanions such as poly(aspartic acid) and hyaluronic acid. This is expected to be essential for in vivo application of antibody-targeted LPP as naturally occurring polyanions have been shown to have detrimental effects on plain polyplexes after intraperitoneal administration. After intravenous administration in mice, p(DMAEMA)/[32P]-pLuc complexes distributed primarily to the lungs. The gene expression profile matched the biodistribution profile. In vitro evidence was collected pointing to aggregate formation and trapping of the formed aggregates in the lung capillary bed as a primary mechanism explaining the dominant lung uptake and transfection. Therefore, it was investigated whether shielding of the surface positive charge of the polyplexes can increase colloidal stability and prevent dominant lung uptake. Recent mice experiments yielded successful results with surface modification of the p(DMAEMA)-based polyplexes with polyethylene glycol (PEG). Prolonged circulation and avoidance of dominant lung localization were observed after intravenous administration of the PEGylated polyplexes. Most importantly, a significant degree of tumor targeting was observed in the subcutaneous C26 colon carcinoma mouse model.

Keywords: dimethylamino)ethyl methacrylate, transfection



149-157

Regulation of globin genes expression: New findings made with the chicken domain of α globin genes

Author(s): Dr. Razin, Sergey,

Abstract: The domain of chicken α globin genes represents one of the best studied genomic domains in higher eukaryotes. Nevertheless, many questions concerning the nature of mechanisms regulating coordinated expression of globin genes in the course of development remain open. Here we show, that the whole cluster of α globin genes is preceded by a CpG-rich region which colocalises with the replication origin and the permanent site of DNA attachment to the nuclear matrix. In non-erythroid cells the upstream CpG-rich area of the α-globin gene domain is selectively methylated. In model experiments, methylation of this sequence element exerted a strong negative effect on the activity of globin gene promoters. We suggest that the upstream CpG rich area of the α-globin gene domain constitute a molecular switch which regulate expression of α globin genes in cells of different lineage. α-globin genes are found to be transcribed in both proliferating (premature) and differentiated (mature) erythroid cells. However, only the latter express globins at a protein level. We found that the block of productive expression of globins in premature erythroid cells occurs at post-transcriptional level. In these cells the transcripts of α-globin genes are retained in nuclei. Induction of proliferating erythroid cells to differentiation is accompanied by a release of globin gene transcripts to the cytoplasm.

Keywords:



149-157

Regulation of globin genes expression: New findings made with the chicken domain of α globin genes

Author(s): Dr. Razin Sergey,

Abstract: The domain of chicken α globin genes represents one of the best studied genomic domains in higher eukaryotes. Nevertheless, many questions concerning the nature of mechanisms regulating coordinated expression of globin genes in the course of development remain open. Here we show, that the whole cluster of α globin genes is preceded by a CpG-rich region which colocalises with the replication origin and the permanent site of DNA attachment to the nuclear matrix. In non-erythroid cells the upstream CpG-rich area of the α-globin gene domain is selectively methylated. In model experiments, methylation of this sequence element exerted a strong negative effect on the activity of globin gene promoters. We suggest that the upstream CpG rich area of the α-globin gene domain constitute a molecular switch which regulate expression of α globin genes in cells of different lineage. α-globin genes are found to be transcribed in both proliferating (premature) and differentiated (mature) erythroid cells. However, only the latter express globins at a protein level. We found that the block of productive expression of globins in premature erythroid cells occurs at post-transcriptional level. In these cells the transcripts of α-globin genes are retained in nuclei. Induction of proliferating erythroid cells to differentiation is accompanied by a release of globin gene transcripts to the cytoplasm.

Keywords: globin genes expression, chicken domain, α-globin genes



159-167

Surface-shielded polycation-based systems targeting reporter and therapeutic genes to distant tumors

Author(s): Dr. Ralf Kircheis,

Abstract: We have developed surface-shielded transferrin-polyethylenimine (Tf-PEI) - based gene delivery systems which are able to target gene expression to distant tumors after systemic application in murine models. For systemic in vivo application the biophysical parameters of transfection complexes, such as particle size, stability, surface charge, and modification with targeting ligand, were found to be critical for DNA biodistribution, toxicity, and gene transfer efficacy. Two major mechanisms may contribute to the tumor-specific targeting: active targeting via receptor- mediated cell binding and passive targeting via shielding of the surface charge of the complexes. Shielding reduces plasma protein and erythrocyte binding, resulting in prolonged blood circulation and extravasation of DNA complexes in areas of vascular leakiness of the tumor tissue. Shielding of surface charges can be achieved by coating polycation/DNA complexes with either polyethylene glycol (PEG) or by incorporating Tf ligand at high densities. Systemic application of surface-shielded transferrin-polyethylenimine-based DNA complexes coding for tumor necrosis factor (TNFα) localized gene expression to distant tumors, resulting in pronounced hemorrhagic tumor necrosis and inhibition of tumor growth. TNFα activity was confined to the tumor without systemic TNF-related toxicity.

Keywords: gene therapy, tumor targeting, non-viral gene transfer, polyethylenimine, PEI, polyplex, transferrin, tumor necrosis factor, TNFα



169-181

A retroviral model for tissue-specific transcription: lessons for gene therapy

Author(s): Dr. Jaquelin P. Dudley,

Abstract: Mouse mammary tumor virus (MMTV) induces breast cancer in mice by transmission of virus from infected mothers to susceptible offspring through milk. During milk-borne MMTV transmission, virus must be transferred to B and T cells in the gut-associated lymphoid tissue, and these lymphocytes carry the infection to the mammary gland. Wild-type MMTV strains have been selected for optimal virus expression in lactating mammary gland cells, while minimizing gene expression and integration in other cell types. In particular, the MMTV transcriptional control region contains binding sites for both transcriptional repressor proteins, e.g., SATB1 and CDP, and positive factors, e.g., glucocorticoid receptor. Studies of MMTV transcriptional regulation may provide important lessons for the design of effective and safe gene therapy vectors.

Keywords: MMTV, T-cell lymphoma, steroid reseptor



183-194

Integrating vector and stem cell-based strategies for gene therapy of Duchenne muscular dystrophy

Author(s): Dr. ,

Abstract: We review novel gene transfer strategies proposed to be suitable for the treatment Duchenne Muscular Dystrophy (DMD): use of hybrid adeno-retroviral vectors to stably replace dystrophin ultimately in patients lacking this gene and the potential intravenous application of stem cells and monocytes for targeted gene transfer. We discuss the limitations of current vector technology and demonstrate the need for continual evolution of vector design that is required before gene therapy of complex monogenic diseases such as DMD becomes a reality.

Keywords: muscular dystrophy, hybrid virus, adenovirus, retrovirus, macrophage, and stem cell



195-200

Bifidobacterium longum as a gene delivery system for cancer gene therapy

Author(s): Dr. Minoru Fujimori,

Abstract: A fundamental obstacle in cancer gene therapy is the specific targeting of therapy directly to a solid tumor, and no systemic delivery system yet exists. A strain of domestic bacteria, Bifidobacterium longum, which is nonpathogenic and anaerobic, selectively localized to and proliferated in solid tumors after systemic application. We propose a novel approach to cancer gene therapy in which anaerobic bacteria of the genus Bifidobacterium longum (B. longum) are used to achieve tumor specific gene delivery and prodrug-enzyme therapy. This is the first demonstration that Bifidobacterium longum can be utilized as a specific gene delivery vector for gene therapy on solid tumors.

Keywords: cancer, prodrug-enzyme therapy




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