High expression of transgenes mediated by hybrid retroviral vectors in hepatocytes: comparison of promoters from murine retroviruses in vitro and in vivo

Ohnishi, Naoki; Itoh, Katsuhiko; Itoh, Yoshito; Baum, Christopher; Higashitsuji, Hiroaki; Yamaguchi, Kanji; Tsuji, Takashi; Okanoue, Takeshi; Fujita, Jun

doi:10.1038/sj.gt.3301655

“…The MSCV retroviral LTR has been shown to drive high expression in hematopoietic and embryonic stem cells 30, 31. The spleen focus‐forming virus LTR induces high levels of expression in hepatocytes and hematopoietic cells 32, 33. Here, we found that this latter LTR used in pFIP/CD promoted higher levels of CD expression in MSCs than did the MSCV‐LTR (Fig.…”

Section: Discussionmentioning

confidence: 57%

Five‐year outcome of patients classified using the American Society for Radiation Oncology consensus statement guidelines for the application of accelerated partial breast irradiation

Shaitelman

¹

,

Vicini

²

,

Beitsch

³

et al. 2010

Cancer

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Suicide genes have recently emerged as an attractive alternative therapy for the treatment of various types of intractable cancers. The efficacy of suicide gene therapy relies on efficient gene delivery to target tissues and the localized concentration of final gene products. Here, we showed a potential ex vivo therapy that used mesenchymal stem cells (MSCs) as cellular vehicles to deliver a bacterial suicide gene, cytosine deaminase (CD) to brain tumors. MSCs were engineered to produce CD enzymes at various levels using different promoters. When co‐cultured, CD‐expressing MSCs had a bystander, anti‐cancer effect on neighboring C6 glioma cells in proportion to the levels of CD enzymes that could convert a nontoxic prodrug, 5‐fluorocytosine (5‐FC) into cytotoxic 5‐fluorouracil (5‐FU) in vitro. Consistent with the in vitro results, for early stage brain tumors induced by intracranial inoculation of C6 cells, transplantation of CD‐expressing MSCs reduced tumor mass in proportion to 5‐FC dosages. However, for later stage, established tumors, a single treatment was insufficient, but only multiple transplantations were able to successfully repress tumor growth. Our findings indicate that the level of total CD enzyme activity is a critical parameter that is likely to affect the clinical efficacy for CD gene therapy. Our results also highlight the potential advantages of autograftable MSCs compared with other types of allogeneic stem cells for the treatment of recurrent glioblastomas through repetitive treatments.

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“…30,31 The spleen focus-forming virus LTR induces high levels of expression in hepatocytes and hematopoietic cells. 32,33 Here, we found that this latter LTR used in pFIP/ CD promoted higher levels of CD expression in MSCs than did the MSCV-LTR (Fig. 1c) and 5-FU production was 4.8-fold higher with pFIP/CD than with pMSCV/CD (Fig.…”

Section: Cancer Therapymentioning

confidence: 60%

The growth of brain tumors can be suppressed by multiple transplantation of mesenchymal stem cells expressing cytosine deaminase

Chang

¹

,

Yoo

²

,

Hong

³

et al. 2010

Intl Journal of Cancer

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Suicide genes have recently emerged as an attractive alternative therapy for the treatment of various types of intractable cancers. The efficacy of suicide gene therapy relies on efficient gene delivery to target tissues and the localized concentration of final gene products. Here, we showed a potential ex vivo therapy that used mesenchymal stem cells (MSCs) as cellular vehicles to deliver a bacterial suicide gene, cytosine deaminase (CD) to brain tumors. MSCs were engineered to produce CD enzymes at various levels using different promoters. When co-cultured, CD-expressing MSCs had a bystander, anti-cancer effect on neighboring C6 glioma cells in proportion to the levels of CD enzymes that could convert a nontoxic prodrug, 5-fluorocytosine (5-FC) into cytotoxic 5-fluorouracil (5-FU) in vitro. Consistent with the in vitro results, for early stage brain tumors induced by intracranial inoculation of C6 cells, transplantation of CD-expressing MSCs reduced tumor mass in proportion to 5-FC dosages. However, for later stage, established tumors, a single treatment was insufficient, but only multiple transplantations were able to successfully repress tumor growth. Our findings indicate that the level of total CD enzyme activity is a critical parameter that is likely to affect the clinical efficacy for CD gene therapy. Our results also highlight the potential advantages of autograftable MSCs compared with other types of allogeneic stem cells for the treatment of recurrent glioblastomas through repetitive treatments.Gliomas are the most common primary tumors of the central nervous system and easily metastasize to other brain regions. Despite recent technical advances in surgery, radiotherapy and chemotherapy, only palliative therapy is available; there is no authentic cure for gliomas. 1,2 Suicide genes from non-human sources have emerged as an attractive alternative therapy for the treatment of brain tumors. Cytosine deaminase (CD) has attracted considerable attention by virtue of its stronger bystander effects than other suicide genes. 3 CD converts a nontoxic prodrug, 5-fluorocytosine (5-FC), into a potent anticancer drug, 5-fluorouracil (5-FU). 5-FU enters neighboring cells through simple diffusion, exerting a cytotoxic effect by interfering with DNA and RNA synthesis. By combining CD and 5-FC in a single therapeutic modality, it may be possible to bypass the systemic toxicity of 5-FU while efficiently increasing the 5-FU concentration in a localized area. 4,5 Neural stem cells (NSCs) and mesenchymal stem cells (MSCs) exhibit strong tropism toward brain tumors that are attributed to receptors for chemokines and growth factors including SDF-1, 6,7 MCP-1, 8 HGF, 9 IL-8, NT3, TGF-b 10 and VEGF. 11 The migratory properties make NSCs and MSCs efficient tools for delivering therapeutic genes to brain tumors. For example, when combined with 5-FC administration,

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“…Having previously observed retroviral silencing in over 95% of MoMLV transduced cells, Prasad Alur et al [Alur et al, 2002] recently demonstrated that deleting negatively acting cis elements in the viral LTRs, retaining a permissive primer binding site (PBS) sequence and using the eukaryotic β-glucuronidase promoter could overcome this downregulation in vivo in a mouse model of lysosomal enzyme deficiency. Another study demonstrated that the use of alternative optimised LTR promoters to drive expression form retroviral vectors may avoid severe silencing effects in the liver in vivo [Ohnishi et al, 2002]. Further mechanisms of avoiding the silencing of viral promoters, such as the use of LCRs and MARs, are discussed later.…”

Section: Retroviral Ltr Promotersmentioning

confidence: 99%

Promoters and Control Elements: Designing Expression Cassettes for Gene Therapy

Papadakis

¹

,

Nicklin²,

Baker³

et al. 2004

CGT

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It has become apparent that the clinical success anticipated in the field of gene therapy has been limited by progress in several of the fundamental areas of genetics, molecular and cellular biology relevant to its application. Whilst a great deal of effort has been made in the evaluation of transgenes, it is only more recently with the advance of vector systems that attention has begun to be focused upon the means and control of transgene expression. Until recently, the majority of constructs have employed ubiquitous viral promoters to drive expression from simple gene expression cassettes using viral promoters and lacking introns, 3' untranslated regions (UTRs), locus control regions (LCR's), matrix attachment regions (MAR's) and other such genetic components. It has consequently emerged that these elements may have a key role in determining the levels and longevity of gene expression attainable in vivo, irrespective of the vector system utilised. The majority of gene therapy applications would also benefit from the specific optimisation of 'tailormade' expression cassettes to optimise their therapeutic efficacy. In conjunction with modification of vector tropism and strategies to limit their immunogenicity, this should create vectors suitable for the clinical application of gene therapy. This review aims to highlight some of the principle considerations of gene expression in vivo, and the means by which it may most effectively be achieved, whether this is via the minimal modification of an existing eukaryotic promoter or by the more extensive design of a novel promoter and associated elements.

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High expression of transgenes mediated by hybrid retroviral vectors in hepatocytes: comparison of promoters from murine retroviruses in vitro and in vivo

Cited by 8 publications

References 23 publications

Five‐year outcome of patients classified using the American Society for Radiation Oncology consensus statement guidelines for the application of accelerated partial breast irradiation

Five‐year outcome of patients classified using the American Society for Radiation Oncology consensus statement guidelines for the application of accelerated partial breast irradiation

The growth of brain tumors can be suppressed by multiple transplantation of mesenchymal stem cells expressing cytosine deaminase

Promoters and Control Elements: Designing Expression Cassettes for Gene Therapy

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