Intratumoral Spread and Increased Efficacy of a p53-VP22 Fusion Protein Expressed by a Recombinant Adenovirus

ABSTRACT In vitro experiments have demonstrated intercellular trafficking of the VP22 tegument protein of herpes simplex virus type 1 from infected cells to neighboring cells, which internalize VP22 and transport it to the nucleus. VP22 also can mediate intercellular transport of fusion proteins, providing a strategy for increasing the distribution of therapeutic proteins in gene therapy. Intercellular trafficking of the p53 tumor suppressor protein was demonstrated in vitro using a plasmid expressing full-length p53 fused in-frame to full-length VP22. The p53-VP22 chimeric protein induced apoptosis both in transfected tumor cells and in neighboring cells, resulting in a widespread cytotoxic effect. To evaluate the anti-tumor activity of p53-VP22 in vivo, we constructed recombinant adenoviruses expressing either wild-type p53 (FTCB) or a p53-VP22 fusion protein (FVCB) and compared their effects in p53-resistant tumor cells. In vitro, treatment of tumor cells with FVCB resulted in enhanced p53-specific apoptosis compared to treatment with equivalent doses of FTCB. However, in normal cells there was no difference in the dose-related cytotoxicity of FVCB compared to that of FTCB. In vivo, treatment of established tumors with FVCB was more effective than equivalent doses of FTCB. The dose-response curve to FVCB was flatter than that to FTCB; maximal antitumor responses could be achieved using FVCB at doses 1 log lower than those obtained with FTCB. Increased antitumor efficacy was correlated with increased distribution of p53 protein in FVCB-treated tumors. This study is the first demonstration that VP22 can enhance the in vivo distribution of therapeutic proteins and improve efficacy in gene therapy.

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Recombinant adenovirus of SEA and CD80 genes driven by MMRE and mouse TERT promoter induce effective antitumor immune responses against different types of tumor cells in vitro and in vivo

Oncology Reports ◽

10.3892/or.2017.5563 ◽

2017 ◽

Vol 37 (5) ◽

pp. 3037-3045

Author(s):

Shao-Yan Si ◽

Jun-Li Liu ◽

Jun-Lian Liu ◽

Bing-Xin Xu ◽

Jian-Zhong Li ◽

...

Keyword(s):

Immune Responses ◽

Tumor Cells ◽

Recombinant Adenovirus ◽

Tert Promoter ◽

Different Types

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ADAMTS13 Binds VWF Via its C-Terminal CUB2 Domain.

Blood ◽

10.1182/blood.v104.11.126.126 ◽

2004 ◽

Vol 104 (11) ◽

pp. 126-126 ◽

Cited By ~ 2

Author(s):

Weirui Zhang ◽

David Motto ◽

David Ginsburg

Keyword(s):

Fusion Protein ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Expression Vector ◽

Full Length ◽

Mammalian Expression ◽

Partial Denaturation

Abstract Thrombotic thrombocytopenic purpura (TTP) is a life threatening illness due to a deficiency of the VWF-cleaving protease, ADAMTS13. The ADAMTS13 protein is composed of a propeptide, followed by a typical zinc metalloprotease domain. The C-terminal 2/3 of the molecule contains disintegrin-like, cystine-rich, and spacer domains, as well as a total of eight TSP1 motifs and two CUB domains. The function of this C-terminal portion of the molecule and its composite motifs is unknown, though TSP1 and CUB domains of other proteins have been shown to mediate protein-protein interactions. To further explore the interaction between ADAMTS13 and VWF, we cloned full length human cDNAs for both ADAMTS13 and VWF into the mammalian expression vector pcDNA3.1. These constructs were transiently transfected into 293T cells and COS cells respectively, and conditioned media collected for analysis. Using an anti-myc antibody, myc-tagged VWF co-immunoprecipitated (co-IP) with ADAMTS13, as demonstrated by western blot analysis using antisera raised against a C-terminal peptide derived from the predicted ADAMTS13 sequence. This direct interaction required partial denaturation of VWF in 1M urea, with no co-IP observed in the absence of urea. To map the segment within ADAMTS13 responsible for VWF binding, we cloned a series of overlapping ADAMTS13 fragments into the bacterial expression vector, Pet44b. Fusion proteins were purified by binding of the included His-tag to Ni-NTA beads and incubated with recombinant myc-VWF in the presence of 1M urea. Association with VWF was analyzed by co-IP with anti-myc followed by western blot analysis using an antibody to the C-terminal HSV-tag present in each fusion protein. The CUB2 (Glu1298- Thr1427) fusion protein co-IP’d with full-length VWF and also demonstrated concentration-dependent competition with full-length ADAMTS13 for VWF binding. In summary, we have demonstrated a direct protein-protein interaction between VWF and ADAMTS13. Binding requires partial denaturation of VWF and appears to be mediated primarily through contacts with the ADAMTS13 CUB2 domain. This interaction may account for the previously observed co-purification of VWF and ADAMTS13 from human plasma. Furthermore, the requirement for 1M urea suggests that this interaction may only occur physiologically under conditions of high shear. Though others have shown that the C-terminal domains of ADAMTS13, including CUB2, are not required for VWF cleavage in vitro, our data, together with several C-terminal mutations previously reported in TTP patients, suggest that interactions between VWF and the ADAMTS13 CUB2 domain may be important in vivo.

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Adenovirus-Mediated Expression of a Ribozyme to c-mybmRNA Inhibits Smooth Muscle Cell Proliferation and Neointima Formation In Vivo

Journal of Virology ◽

10.1128/jvi.73.9.7745-7751.1999 ◽

1999 ◽

Vol 73 (9) ◽

pp. 7745-7751 ◽

Cited By ~ 30

Author(s):

Dennis G. Macejak ◽

Hua Lin ◽

Saiphone Webb ◽

Jennifer Chase ◽

Kristi Jensen ◽

...

Keyword(s):

Gene Therapy ◽

Smooth Muscle ◽

Animal Models ◽

Smooth Muscle Cell ◽

Muscle Cell ◽

Recombinant Adenovirus ◽

Neointima Formation ◽

Inhibition Of Proliferation

ABSTRACT Smooth muscle cell (SMC) proliferation is an important component of restenosis in response to injury after balloon angioplasty. Inhibition of proliferation in vivo can limit neointima hyperplasia in animal models of restenosis. Ribozymes against c-myb mRNA have been shown to be effective inhibitors of SMC proliferation in vitro. The effectiveness of adenovirus as a gene therapy vector in animal models of restenosis is well documented. In order to test the utility of ribozymes to inhibit SMC proliferation by a gene therapy approach, recombinant adenovirus expressing ribozymes against c-mybmRNA was generated and tested both in vitro and in vivo. This adenovirus ribozyme vector is shown to inhibit SMC proliferation in culture and neointima formation in a rat carotid artery balloon injury model of restenosis.

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Enhancing the antitumor functions of invariant natural killer T cells using a soluble CD1d-CD19 fusion protein

Blood Advances ◽

10.1182/bloodadvances.2018028886 ◽

2019 ◽

Vol 3 (5) ◽

pp. 813-824 ◽

Cited By ~ 1

Author(s):

Rupali Das ◽

Peng Guan ◽

Susan J. Wiener ◽

Nishant P. Patel ◽

Trevor G. Gohl ◽

...

Keyword(s):

Fusion Protein ◽

B Cell ◽

Natural Killer ◽

Tumor Cells ◽

Inkt Cell ◽

Inkt Cells ◽

Cell Responses ◽

Invariant Natural Killer

Abstract Invariant natural killer T (iNKT) cells comprise a unique lineage of CD1d-restricted lipid-reactive T lymphocytes that potently kill tumor cells and exhibit robust immunostimulatory functions. Optimal tumor-directed iNKT cell responses often require expression of the antigen-presenting molecule CD1d on tumors; however, many tumor cells downregulate CD1d and thus evade iNKT cell recognition. We generated a soluble bispecific fusion protein designed to direct iNKT cells to the site of B-cell cancers in a tumor antigen-specific but CD1d-independent manner. This fusion protein is composed of a human CD1d molecule joined to a single chain antibody FV fragment specific for CD19, an antigen widely expressed on B-cell cancers. The CD1d-CD19 fusion protein binds specifically to CD19-expressing, but not CD19-negative cells. Once loaded with the iNKT cell lipid agonist α-galactosyl ceramide (αGC), the CD1d-CD19 fusion induces robust in vitro activation of and cytokine production by human iNKT cells. iNKT cells stimulated by the αGC-loaded CD1d-CD19 fusion also strongly transactivate T-, B-, and NK-cell responses and promote dendritic cell maturation. Importantly, the αGC-loaded fusion induces robust lysis of CD19+CD1d− Epstein-Barr virus immortalized human B-lymphoblastoid cell lines that are otherwise resistant to iNKT cell killing. Consistent with these findings; administration of the αGC-loaded fusion protein controlled the growth of CD19+CD1d− tumors in vivo, suggesting that it can “link” iNKT cells and CD19+CD1d− targets in a therapeutically beneficial manner. Taken together, these preclinical studies demonstrate that this B cell–directed fusion protein can be used to effectively induce iNKT cell antitumor responses in vitro and in vivo.

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P28-Fur-Grb, A Novel Fusion Protein with Enhanced Targeted Cytotoxicity Against Breast Cancer

Clinical Oncology and Research ◽

10.31487/j.cor.2019.05.02 ◽

2019 ◽

pp. 1-8

Author(s):

Saeed Ranjbar ◽

Aria Momeni ◽

Azadeh Reshadmanesh ◽

Azita Fakhravar ◽

Nafiseh Paydarnia ◽

...

Keyword(s):

Breast Cancer ◽

Fusion Protein ◽

Tumor Cells ◽

Breast Cancer Cell Lines ◽

Target Cells ◽

Proliferative Potential ◽

Furin Cleavage ◽

Ic50 Values

Targeting tumor cells via multiple pathways promises the emergence of a new era in cancer therapy. Consisting of a cell-binding ligand and a cytotoxic moiety, cytolytic fusion proteins can selectively bind and kill target cells with minimal adverse effects. We designed a novel immunoproapoptotic fusion protein, p28-fur-GrB, composed of the cancer-specific azurin-derived cell penetrating peptide, p28, and a mutant version of human serine protease granzyme B. The two moieties were genetically fused by a furin sensitive linker, allowing in vivo cleavage and activation of the immunotoxin after cell entry. Synthesized coding gene of the recombinant protein was cloned and expressed in HEK293T cells, and nickel chromatography was applied for protein purification. After in vitro furin cleavage and primary analyses of SDS-PAGE, Western blotting, GrB activity and ELISA binding assay, the fusion protein was tested for its cytotoxicity on various breast cancer cell lines. Suppression of cell proliferation and viability was evaluated using the WST-1 assay. Furthermore, DNA fragmentation was measured as an indication of apoptotic effects of the fusion protein on treated cells. Based on our results, p28-fur-GrB was efficiently cleaved by furin and showed high GrB activity and binding affinity after cleavage. Following 72h of incubation with IC50 values of the fusion protein, significant cytotoxic effects of 80.6%, 77.1%, 74% and 69.6% were recorded for BT474, MCF7, SK-BR-3 and MDA-MB-231 tumor cells, respectively. Proliferative potential of MCF 10A normal cells was not affected by the treatment. Analysis of the rate of apoptosis in treated cells confirmed our cytotoxicity results. We concluded that p28-fur-GrB is a potent anti-tumor agent with high cytotoxicity against breast cancer cells.

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IGF-I: from diagnostic to triple-helix gene therapy of solid tumors.

Acta Biochimica Polonica ◽

10.18388/abp.2002_3757 ◽

2002 ◽

Vol 49 (4) ◽

pp. 979-990 ◽

Cited By ~ 12

Author(s):

Ladislas A Trojan ◽

Piotr Kopinski ◽

Ming X Wei ◽

Adama Ly ◽

Aleksandra Glogowska ◽

...

Keyword(s):

Gene Therapy ◽

Growth Factor ◽

Solid Tumors ◽

Tumor Cells ◽

Triple Helix ◽

Human Tumor ◽

Igf I ◽

I Gene

Alterations in the expression of growth factors and their receptors are associated with the growth and development of human tumors. One such growth factor is IGF-I (insulin-like growth factor I ), a 70-amino-acid polypeptide expressed in many tissues, including brain. IGF-I is also expressed at high levels in some nervous system-derived tumors, especially in glioblastoma. When using IGF-I as a diagnostic marker, 17 different tumors are considered as expressing the IGF-I gene. Malignant glioma, the most common human brain cancer, is usually fatal. Average survival is less than one year. Our strategy of gene therapy for the treatment of gliomas and other solid tumors is based on: 1) diagnostic using IGF-I gene expression as a differential marker, and 2) application of "triple-helix anti-IGF-I" therapy. In the latter approach, tumor cells are transfected with a vector, which encodes an oligoribonucleotide--an RNA strand containing oligopurine sequence which might be capable of forming a triple helix with an oligopurine and/or oligopyrimidine sequence of the promotor of IGF-I gene (RNA-IGF-I DNA triple helix). Human tumor cells transfected in vitro become down-regulated in the production of IGF-I and present immunogenic (MHC-I and B7 expression) and apoptotic characteristics. Similar results were obtained when IGF-I antisense strategy was applied. In both strategies the transfected cells reimplanted in vivo lose tumorigenicity and elicit tumor specific immunity which leads to elimination of established tumors.

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Inhibition of breast cancer cells by targeting E2F-1 gene and expressing IL15 oncolytic adenovirus

Bioscience Reports ◽

10.1042/bsr20190384 ◽

2019 ◽

Vol 39 (7) ◽

Cited By ~ 3

Author(s):

Yang Yan ◽

Hu Xu ◽

Jiandong Wang ◽

Xin Wu ◽

Wei Wen ◽

...

Keyword(s):

Breast Cancer ◽

Gene Therapy ◽

Recombinant Adenovirus ◽

Oncolytic Adenovirus ◽

The Novel ◽

Gene Therapy Application ◽

Interleukin 15 ◽

Breast Cancer Virus

Abstract The wide application of oncolytic adenovirus presents a novel therapeutic strategy for breast cancer gene therapy. Application of adenovirus alone achieves little curative effects on breast cancer. In addition, it is worth exploring the synergistic anti-tumor effect by inserting immunomodulatory factor in oncolytic adenovirus genome. By taking the advantage of the highly proliferative property of breast cancer, a novel recombinant adenovirus which could selectively kill tumor cells is established under an E2F-1 promoter. Also by carrying human Interleukin-15 (IL-15) gene, the oncolytic adenovirus exhibits an immunomodulatory effect. The present study proved that the novel oncolytic virus (SG400-E2F/IL-15) exhibits an enhanced anti-tumor activity both in vitro and in vivo, representing an experimental basis for breast cancer “virus-gene” therapy.

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Oncolytic adenovirus and gene therapy with EphA2-BiTE for the treatment of pediatric high-grade gliomas

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001930 ◽

2021 ◽

Vol 9 (5) ◽

pp. e001930

Author(s):

Claudia Manuela Arnone ◽

Vinicia Assunta Polito ◽

Angela Mastronuzzi ◽

Andrea Carai ◽

Francesca Camassei Diomedi ◽

...

Keyword(s):

Gene Therapy ◽

T Cells ◽

T Cell ◽

Tumor Cells ◽

Tumor Control ◽

Malignant Neoplasms ◽

High Grade ◽

High Grade Gliomas

BackgroundPediatric high-grade gliomas (pHGGs) are among the most common and incurable malignant neoplasms of childhood. Despite aggressive, multimodal treatment, the outcome of children with high-grade gliomas has not significantly improved over the past decades, prompting the development of innovative approaches.MethodsTo develop an effective treatment, we aimed at improving the suboptimal antitumor efficacy of oncolytic adenoviruses (OAs) by testing the combination with a gene-therapy approach using a bispecific T-cell engager (BiTE) directed towards the erythropoietin-producing human hepatocellular carcinoma A2 receptor (EphA2), conveyed by a replication-incompetent adenoviral vector (EphA2 adenovirus (EAd)). The combinatorial approach was tested in vitro, in vivo and thoroughly characterized at a molecular level.ResultsAfter confirming the relevance of EphA2 as target in pHGGs, documenting a significant correlation with worse clinical outcome of the patients, we showed that the proposed strategy provides significant EphA2-BiTE amplification and enhanced tumor cell apoptosis, on coculture with T cells. Moreover, T-cell activation through an agonistic anti-CD28 antibody further increased the activation/proliferation profiles and functional response against infected tumor cells, inducing eradication of highly resistant, primary pHGG cells. The gene-expression analysis of tumor cells and T cells, after coculture, revealed the importance of both EphA2-BiTE and costimulation in the proposed system. These in vitro observations translated into significant tumor control in vivo, in both subcutaneous and a more challenging orthotopic model.ConclusionsThe combination of OA and EphA2-BiTE gene therapy strongly enhances the antitumor activity of OA, inducing the eradication of highly resistant tumor cells, thus supporting the clinical translation of the approach.

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Suicide gene therapy against cancer

ACTUALIDAD MEDICA ◽

10.15568/am.2021.812.rev01 ◽

2021 ◽

Vol 106 (106(812)) ◽

pp. 54-65

Author(s):

C. Piñeiro-Silva

Keyword(s):

Gene Therapy ◽

Tumor Cells ◽

Large Family ◽

Suicide Gene ◽

Suicide Gene Therapy ◽

Gene Encoding ◽

Bacterial Enzyme ◽

Proapoptotic Gene

Cancers are a large family of diseases with the highest mortality rate worldwide. Conventional therapies such as chemotherapy or radiotherapy are not efficient in all cases and have important side effects. To solve it, suicide gene therapy can be used. This therapy consists on inducing cell death of cancer cells due to the introduction of a gene. There are three types of this therapy: introduction of a gene encoding generally a bacterial enzyme that actives a prodrug, introduction of a gene encoding a toxin or introduction of a proapoptotic gene. The expression of the targeted gene in tumor cells is produced by using tumor-specific promoters and target vectors. Using those three gene suicide therapies many hallmarks in the field were reached, achieving successful clinical trials and products approved to be used in China (gendicine), achieving apoptosis of tumor cells in vitro and in vivo. Furthermore, several improvements on these techniques were developed due to the mutation of the enzymes and toxins, modification of prodrugs and search of new more active enzymes, toxins and genes, between others. Regardless, further research on this area is needed to guarantee the efficiency of this state-of-the-art therapy and its effectiveness.

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