Limited efficacy of gene transfer in herpes simplex virus-thymidine kinase/ganciclovir gene therapy for brain tumors

2005 ◽  
Vol 102 (2) ◽  
pp. 328-335 ◽  
Author(s):  
Piotr Hadaczek ◽  
Hanna Mirek ◽  
Mitchel S. Berger ◽  
Krystof Bankiewicz
Keyword(s):  
Gene Therapy ◽  
Brain Tumor ◽  
Gene Transfer ◽  
Thymidine Kinase ◽  
Therapeutic Effects ◽  
Content Type ◽  
Over Time ◽  
Fine Print

Object. Low efficacy of gene transfer, transient gene expression, and toxicity of viral vectors are the major hurdles in successful anticancer gene therapy. The authors conducted in vitro (U87MG cell line) and in vivo (xenograft, tumor-bearing rodent model) studies to address the stability of transduction by using the adenoassociated virus serotype-2 (AAV2)—thymidine kinase (TK) vector over time. Methods. Standard methods for cell growth and a ganciclovir (GCV) cytotoxicity assay were applied. The AAV2-TK was infused into implanted tumors in athymic rats via convection-enhanced delivery (CED). Thymidine kinase expression was evaluated through immunohistochemical analysis, and the distribution volumes of the transduced tumors were calculated. Twenty-four hours following the viral infusions, animals were treated with GCV (50 mg/kg intraperitoneally every day for 10 days; six rats) or phosphate-buffered saline (six rats). A rapid decrease in TK expression over time was observed both in vitro and in vivo. A large volume of the tumor (up to 39%) was transduced with AAV2-TK following CED. Administration of GCV resulted in limited therapeutic effects (survival of 25.8 compared with 21.3 days). Conclusions. Rapid elimination of TK expression from dividing tumor cells and focal transduction of the brain tumor were most likely responsible for the limited bystander effect in this approach. Immediate administration of GCV is crucial to assure maximal efficacy in the elimination of cancer cells. In addition, the complete or diffused transduction of a brain tumor with TK may be required for its total eradication.

Assessment of brain tumor cell motility in vivo and in vitro

1995 ◽  
Vol 82 (4) ◽  
pp. 615-622 ◽  
Author(s):  
Michael R. Chicoine ◽  
Daniel L. Silbergeld
Keyword(s):  
Brain Tumor ◽  
Cell Motility ◽  
Human Brain Tumor ◽  
Content Type ◽  
Brain Tumor Cells ◽  
Tumor Cell Motility ◽  
Brain Tumor Cell ◽  
Fine Print

✓ Brain tumor dispersal far from bulk tumor contributes to and, in some instances, dominates disease progression. Three methods were used to characterize brain tumor cell motility in vivo and in vitro: 1) 2 weeks after implantation in rat cerebral cortex, single C6 cells labeled with a fluorescent tag had migrated to brain sites greater than 16 mm distant from bulk tumor; 2) time-lapse videomicroscopy of human brain tumor cells revealed motility of 12.5 µm/hr. Ruffling leading edges and pseudopod formation were most elaborate in more malignant cells; 3) an in vitro assay was devised to quantitatively evaluate motility from a region of high cell density to one of lower cell density. Human brain tumor cells were plated in the center of a petri dish, washed, and refed, establishing a 2-cm circular zone of cells in the dish center. Motility was determined by counting cells daily at predetermined distances from the central zone perimeter. Cells were found 1 cm from the perimeter by 24 hours and 3 cm from the perimeter by 4 days. Increasing serum concentration increased motility; however, neither fibronectin nor arrest of cells in the G0 phase by hydroxyurea altered motility. The addition of cytochalasin B to block cytoskeletal assembly prevented cell motility. Motility increased with increased malignancy. Subpopulations of cells were created by clonal amplification of cells that had migrated most rapidly to the dish periphery. Although morphologically indistinguishable when compared to the original cell line from which they were derived, these subpopulations demonstrated significantly increased motility.

Use of bone morphogenetic protein—9 gene therapy to induce spinal arthrodesis in the rodent

2000 ◽  
Vol 92 (2) ◽  
pp. 191-196 ◽  
Author(s):  
Gregory A. Helm ◽  
Tord D. Alden ◽  
Elisa J. Beres ◽  
Sarah B. Hudson ◽  
Subinoy Das ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Morphogenetic Proteins ◽  
Ex Vivo ◽  
Content Type ◽  
Ex Vivo Gene Therapy ◽  
Bone Morphogenetic Protein 9 ◽  
Spinal Arthrodesis ◽  
Fine Print

Object. Bone morphogenetic proteins (BMPs) have been shown to have significant osteoinductive activity in numerous in vitro and in vivo assay systems, and BMP-2 and BMP-7 are currently being evaluated in human clinical studies. In the spinal region, BMPs have been shown to promote spinal arthrodesis at a higher rate than autologous bone alone. The delivery of BMPs via direct or ex vivo gene therapy techniques is also currently being evaluated and has shown promise in several mammalian models. The present study was designed to evaluate the efficacy of the use of direct, percutaneous BMP-9 adenoviral gene therapy to promote spinal fusion in the rodent. Methods. Each animal was injected with 7.5 × 108 pfu of a BMP-9 adenoviral vector in the lumbar paraspinal musculature and allowed to survive 16 weeks. Computerized tomography studies and histological analysis demonstrated massive bone induction at the injection sites, clearly leading to solid spinal arthrodesis, without evidence of pseudarthroses, nerve root compression, or systemic side effects. Conclusions. The results of this study strongly support the advancement of BMP gene therapy techniques toward clinical use.

Antitumor effects of antiprogesterones on human meningioma cells in vitro and in vivo

1994 ◽  
Vol 80 (3) ◽  
pp. 527-534 ◽  
Author(s):  
Yasuhiro Matsuda ◽  
Keiichi Kawamoto ◽  
Katsuzo Kiya ◽  
Kaoru Kurisu ◽  
Kazuhiko Sugiyama ◽  
...  
Keyword(s):  
Marked Reduction ◽  
Tumor Volume ◽  
Collagen Gel ◽  
Antitumor Effects ◽  
Histological Changes ◽  
Content Type ◽  
The Relationship ◽  
Fine Print

✓ The presence of the progesterone receptor (PR) in meningioma tissue has been confirmed by previous investigations. Studies have shown that the antiprogesterone drug, mifepristone, is a potent agent that inhibits the growth of cultured meningioma cells and reduces the size of meningiomas in experimental animal models and humans. However, these studies have not fully examined the relationship between the antitumor effects of an antiprogesterone agent and the expression of the PR. The present study examined the antitumor effects of mifepristone and a new potent antiprogesterone agent, onapristone; a correlation between the antitumor effects of these antiprogesterones and the presence of PR's in meningiomas in vitro and in vivo was also investigated. Meningioma tissue surgically removed from 13 patients was used in this study. In the in vitro arm of the study, mifepristone and onapristone exhibited cytostatic and cytocidal effects against cultured meningioma cells, regardless of the presence or absence of PR's; however, three PR-negative meningiomas showed no response to any dose of mifepristone and/or onapristone. In the in vivo arm, meningioma cells, embedded in a collagen gel, were implanted into the renal capsules of nude mice. Antiprogesterone treatment resulted in a marked reduction of the tumor volume regardless of the presence or absence of PR's. No histological changes in the meningioma cells suggestive of necrosis or apoptosis were detected in any of the mice treated with antiprogesterones. These findings suggest that mifepristone and onapristone have an antitumor effect against meningioma cells via the PR's and/or another receptor, such as the glucocorticoid receptor.

scholarly journals Dissecting the Structure-Function Relationship of a Fungicidal Peptide Derived from the Constant Region of Human Immunoglobulins

2016 ◽  
Vol 60 (4) ◽  
pp. 2435-2442 ◽  
Author(s):  
Tecla Ciociola ◽  
Thelma A. Pertinhez ◽  
Laura Giovati ◽  
Martina Sperindè ◽  
Walter Magliani ◽  
...  
Keyword(s):  
Self Assembly ◽  
Galleria Mellonella ◽  
Critical Role ◽  
Yeast Cells ◽  
Therapeutic Effects ◽  
Constant Region ◽  
Content Type ◽  
Complementarity Determining Regions

ABSTRACTSynthetic peptides encompassing sequences related to the complementarity-determining regions of antibodies or derived from their constant region (Fc peptides) were proven to exert differential antimicrobial, antiviral, antitumor, and/or immunomodulatory activitiesin vitroand/orin vivo, regardless of the specificity and isotype of the parental antibody. Alanine substitution derivatives of these peptides exhibited unaltered, increased, or decreased candidacidal activitiesin vitro. The bioactive IgG-derived Fc N10K peptide (NQVSLTCLVK) spontaneously self-assembles, a feature previously recognized as relevant for the therapeutic activity of another antibody-derived peptide. We evaluated the contribution of each residue to the peptide self-assembling capability by circular-dichroism spectroscopy. The interaction of the N10K peptide and its derivatives withCandida albicanscells was studied by confocal, transmission, and scanning electron microscopy. The apoptosis and autophagy induction profiles in yeast cells treated with the peptides were evaluated by flow cytometry, and the therapeutic efficacy against candidal infection was studied in aGalleria mellonellamodel. Overall, the results indicate a critical role for some residues in the self-assembly process and a correlation of that capability with the candidacidal activities of the peptidesin vitroand their therapeutic effectsin vivo.

scholarly journals Imaging Expression of Cytosine Deaminase-Herpes Virus Thymidine Kinase Fusion Gene (CD/TK) Expression with [124I]FIAU and PET

2002 ◽  
Vol 1 (1) ◽  
pp. 153535002002000 ◽  
Author(s):  
Trevor Hackman ◽  
Michail Doubrovin ◽  
Julius Balatoni ◽  
Tatiana Beresten ◽  
Vladimir Ponomarev ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Thymidine Kinase ◽  
Enzyme Assay ◽  
Fusion Gene ◽  
Cytosine Deaminase ◽  
Prodrug Activation ◽  
Wide Range ◽  
Different Levels

Double prodrug activation gene therapy using the Escherichia coli cytosine deaminase (CD)herpes simplex virus type 1 thymidine kinase ( HSV1-tk) fusion gene ( CD/TK) with 5-fluorocytosine (5FC), ganciclovir (GCV), and radiotherapy is currently under evaluation for treatment of different tumors. We assessed the efficacy of noninvasive imaging with [124I]FIAU (2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-iodo-uracil) and positron emission tomography (PET) for monitoring expression of the CD/TK fusion gene. Walker-256 tumor cells were transduced with a retroviral vector bearing the CD/TK gene (W256CD/TK cells). The activity of HSV1-TK and CD subunits of the CD/TK gene product was assessed in different single cell-derived clones of W256CD/TK cells using the FIAU radiotracer accumulation assay in cells and a CD enzyme assay in cell homogenates, respectively. A linear relationship was observed between the levels of CD and HSV1-tk subunit expression in corresponding clones in vitro over a wide range of CD/TK expression levels. Several clones of W256CD/TK cells with significantly different levels of CD/TK expression were selected and used to produce multiple subcutaneous tumors in rats. PET imaging of HSV1-TK subunit activity with [124I]FIAU was performed on these animals and demonstrated that different levels of CD/TK expression in subcutaneous W256CD/TK tumors can be imaged quantitatively. CD expression in subcutaneous tumor sample homogenates was measured using a CD enzyme assay. A comparison of CD and HSV1-TK subunit enzymatic activity of the CD/TK fusion protein in vivo showed a significant correlation. Knowing this relationship, the parametric images of CD subunit activity were generated. Imaging with [124I]FIAU and PET could provide pre- and posttreatment assessments of CD/TK-based double prodrug activation in clinical gene therapy trials.

Treatment of progressive or recurrent pediatric malignant supratentorial brain tumors with herpes simplex virus thymidine kinase gene vector—producer cells followed by intravenous ganciclovir administration

2000 ◽  
Vol 92 (2) ◽  
pp. 249-254 ◽  
Author(s):  
Roger J. Packer ◽  
Cory Raffel ◽  
Judith G. Villablanca ◽  
Jörg-Christian Tonn ◽  
Stefan E. Burdach ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Herpes Simplex ◽  
Brain Tumors ◽  
Phase I Study ◽  
Retroviral Vector ◽  
Content Type ◽  
Supratentorial Brain Tumors ◽  
Simplex Virus ◽  
Fine Print

Object. The outcome for children with recurrent malignant brain tumors is poor. The majority of patients die of progressive disease within months of relapse, and other therapeutic options are needed. The goal of this Phase I study was to evaluate the safety of in vivo suicide gene therapy in 12 children with recurrent, malignant, supratentorial brain tumors.Methods. After optimal repeated tumor resection, multiple injections of murine vector—producing cells shedding murine replication—defective retroviral vectors coding the herpes simplex virus thymidine kinase type 1 (HSV-Tk1) gene were made into the rim of the resection cavity. Fourteen days after the vector-producing cells were injected, ganciclovir was administered for 14 days. The retroviral vector that was used only integrated and expressed HSV-Tk1 in proliferating cells, which are killed after a series of metabolic events lead to cell death. The median age of the patients was 11 years (range 2–15 years). Treated brain tumors included seven malignant gliomas, two ependymomas, and three primitive neuroectodermal tumors. The patients were treated with one of three escalating dose concentrations of vector-producer cells. Four transient central nervous system adverse effects were considered possibly related to the vector-producing cells. In no child did permanent neurological worsening or ventricular irritation develop, and tests for replication-competent retroviruses yielded negative findings.Conclusions. This Phase I study demonstrates that in vivo gene therapy in which a replication-defective retroviral vector in murine vector—producing cells is delivered by brain injections can be performed with satisfactory safety in a select group of children with localized supratentorial brain tumors.

Clonal Analyses of Retroviral Vector Integrations in ADA-SCID Patients Treated with Stem Cell Gene Therapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3249-3249
Author(s):  
Barbara Cassani ◽  
Grazia Andolfi ◽  
Massimiliano Mirolo ◽  
Luca Biasco ◽  
Alessandra Recchia ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Human Genome ◽  
Ex Vivo ◽  
Cd34 Cells

Abstract Gene transfer into hematopoietic stem/progenitor cells (HSC) by gammaretroviral vectors is an effective treatment for patients affected by severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA)-deficiency. Recent studied have indicated that gammaretroviral vectors integrate in a non-random fashion in their host genome, but there is still limited information on the distribution of retroviral insertion sites (RIS) in human long-term reconstituting HSC following therapeutic gene transfer. We performed a genome-wide analysis of RIS in transduced bone marrow-derived CD34+ cells before transplantation (in vitro) and in hematopoietic cell subsets (ex vivo) from five ADA-SCID patients treated with gene therapy combined to low-dose busulfan. Vector-genome junctions were cloned by inverse or linker-mediated PCR, sequenced, mapped onto the human genome, and compared to a library of randomly cloned human genome fragments or to the expected distribution for the NCBI annotation. Both in vitro (n=212) and ex vivo (n=496) RIS showed a non-random distribution, with strong preference for a 5-kb window around transcription start sites (23.6% and 28.8%, respectively) and for gene-dense regions. Integrations occurring inside the transcribed portion of a RefSeq genes were more represented in vitro than ex vivo (50.9 vs 41.3%), while RIS <30kb upstream from the start site were more frequent in the ex vivo sample (25.6% vs 19.4%). Among recurrently hit loci (n=50), LMO2 was the most represented, with one integration cloned from pre-infusion CD34+ cells and five from post-gene therapy samples (2 in granulocytes, 3 in T cells). Clone-specific Q-PCR showed no in vivo expansion of LMO2-carrying clones while LMO2 gene overexpression at the bulk level was excluded by RT-PCR. Gene expression profiling revealed a preference for integration into genes transcriptionally active in CD34+ cells at the time of transduction as well as genes expressed in T cells. Functional clustering analysis of genes hit by retroviral vectors in pre- and post-transplant cells showed no in vivo skewing towards genes controlling self-renewal or survival of HSC (i.e. cell cycle, transcription, signal transduction). Clonal analysis of long-term repopulating cells (>=6 months) revealed a high number of distinct RIS (range 42–121) in the T-cell compartment, in agreement with the complexity of the T-cell repertoire, while fewer RIS were retrieved from granulocytes. The presence of shared integrants among multiple lineages confirmed that the gene transfer protocol was adequate to allow stable engraftment of multipotent HSC. Taken together, our data show that transplantation of ADA-transduced HSC does not result in skewing or expansion of malignant clones in vivo, despite the occurrence of insertions near potentially oncogenic genomic sites. These results, combined to the relatively long-term follow-up of patients, indicate that retroviral-mediated gene transfer for ADA-SCID has a favorable safety profile.

Phenotypic Correction of Adamts13-Deficient Mice by Early Intra-Amniotic Gene Transfer of Lentiviral Vector Encoding ADAMTS13 Genes.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 197-197
Author(s):  
Masami Niiya ◽  
Masayuki Endo ◽  
Philip W. Zoltick ◽  
Nidal E. Muvarak ◽  
David G. Motto ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Proteolytic Activity ◽  
Lentiviral Vector ◽  
Wild Type ◽  
Occlusion Time ◽  
Carboxyl Terminal ◽  
Human Wild Type

Abstract ADAMTS13, a member of A Disintegrin and Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS) family, is mainly synthesized in the hepatic stellate cells, endothelial cells and megakaryocytes or platelets. It controls the sizes of von Willebrand factor (VWF) multimers by cleaving VWF at the Tyr1605-Met1606 bond. Genetic deficiency of plasma ADAMTS13 activity results in hereditary thrombotic thrombocytopenic purpura (TTP), also named Upshaw-Schülman syndrome. To develop a potential gene therapy approach and to determine the domains of ADAMTS13 required for recognition and cleavage of VWF in vivo, a self-inactivating lentiviral vector encoding human wild-type ADAMTS13 or variant truncated after the spacer domain (construct MDTCS) was administrated by intra-amniotic injection on embryonic day 8. Direct stereomicroscopy and immunofluorescent microscopic analysis revealed that the green fluorescent protein (GFP) reporter, ADAMTS13 and MDTCS were predominantly expressed in the heart, kidneys and skin. The synthesized ADAMTS13 and truncated variant were detectable in mouse plasma by immunoprecipitation and Western blot, as well as by proteolytic cleavage of FRETS-VWF73 substrate. The levels of proteolytic activity in plasma of mice expressing ADAMTS13 and MDTCS were 5 ± 7% and 60 ± 70%, respectively using normal human plasma as a standard, and this proteolytic activity persisted for at least 24 weeks in Adamts13−/−mice and 42 weeks in wild-type mice tested (the duration of observation). The mice expressing both recombinant ADAMTS13 and MDTCS showed a significantly decreased ratio of plasma VWF collagen-binding activity to antigen and a reduction in VWF multimer sizes as compared to those in the controls. Moreover, the mice expressing ADAMTS13 and MDTCS showed a significant prolongation of ferric chloride-induced carotid arterial occlusion time (9.0 ± 0.6 and 25.2 ± 3.2 min, respectively) as compared to the Adamts13−/− mice expressing GFP alone (5.6 ± 0.5 min) (p&lt;0.01). The ferric chloride-induced carotid occlusion time in Adamts13−/− mice expressing ADAMTS13 was almost identical to that in wild type mice with same genetic background (C56BL/6) (8.0 ± 0.2 min) (p&gt;0.05). The data demonstrate the correction of the prothrombotic phenotype in Adamts13−/−mice by gene transfer to the fetus by viral vectors encoding human wild type ADAMTS13 and the carboxyl terminal truncated variant (MDTCS), supporting the feasibility of developing a gene therapy based treatment for hereditary TTP. The discrepancy in the proteolytic activity of MDTCS between in vitro (Zhang P et al. Blood, 2007 in press) and in vivo in the present study suggests the potential cofactors in murine circulation that may rescue the defective proteolytic activity of the carboxyl-terminal truncated ADAMTS13 protease seen in vitro.

Neonatal or hepatocyte growth factor–potentiated adult gene therapy with a retroviral vector results in therapeutic levels of canine factor IX for hemophilia B

Blood ◽  
2003 ◽  
Vol 101 (10) ◽  
pp. 3924-3932 ◽  
Author(s):  
Lingfei Xu ◽  
Cuihua Gao ◽  
Mark S. Sands ◽  
Shi-Rong Cai ◽  
Timothy C. Nichols ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Growth Factor ◽  
Gene Transfer ◽  
Hepatocyte Growth Factor ◽  
Retroviral Vector ◽  
Factor Ix ◽  
Hemophilia B ◽  
Hepatocyte Growth

AbstractHemophilia B is a bleeding disorder resulting from factor IX (FIX) deficiency that might be treated with gene therapy. Neonatal delivery would correct the disease sooner than would transfer into adults, and could reduce immunological responses. Neonatal mice were injected intravenously with a Moloney murine leukemia virus–based retroviral vector (RV) expressing canine FIX (cFIX). They achieved 150% to 280% of normal cFIX antigen levels in plasma (100% is 5 μg/mL), which was functional in vitro and in vivo. Three newborn hemophilia B dogs that were injected intravenously with RV achieved 12% to 36% of normal cFIX antigen levels, which improved coagulation tests. Only one mild bleed has occurred during 14 total months of evaluation. This is the first demonstration of prolonged expression after neonatal gene therapy for hemophilia B in mice or dogs. Most animals failed to make antibodies to cFIX, demonstrating that neonatal gene transfer may induce tolerance. Although hepatocytes from newborns replicate, those from adults do not. Adult mice therefore received hepatocyte growth factor to induce hepatocyte replication prior to intravenous injection of RV. This resulted in expression of 35% of normal cFIX antigen levels for 11 months, although all mice produced anti-cFIX antibodies. This is the first demonstration that high levels of FIX activity can be achieved with an RV in adults without a partial hepatectomy to induce hepatocyte replication. We conclude that RV-mediated hepatic gene therapy is effective for treating hemophilia B in mice and dogs, although the immune system may complicate gene transfer in adults.

Sign in / Sign up

Export Citation Format

Share Document