Empirical advantages of adeno associated viral vectors in vivo gene therapy for arthritis

OBJECTIVE: To evaluate the utility of the adeno associated viral (AAV) vector for gene delivery to joint cells in vivo and in vitro, and to assess its potential as a vector for arthritis gene therapy. METHODS: A recombinant AAV (rAAV) vector expressing the bacterial beta-galactosidase (beta-gal) gene (rAAV-CMV-LacZ) was directly introduced into healthy-normal mouse knees, or arthritic knees in mice overexpressing tumor necrosis factor-alpha (hTNFalpha-Tg). Beta-gal expression levels were determined by immunohistochemistry and chemiluminescence. The transduction efficiency of this vector on primary fibroblast-like synoviocytes (FLS) in vitro was determined by FACS. The effects of UV and gamma-irradiation as well as TNF-alpha on transduction efficiency were determined using the same methods. RESULTS: We found little evidence of rAAV transduction in the joint cells of healthy mice. Target gene expression was detected in all animals at Day 3, and peaked at Day 7 before returning to baseline levels 21 days after injection. In contrast, synoviocytes, articular chondrocytes, and meniscal cells of diseased mice were transduced by rAAV-CMV-LacZ in hTNFalpha-Tg animals. Transduction efficiencies correlated with joint damage, and target gene expression was up to 10-fold greater than that seen in the normal mice. In vitro, we found that rAAV transduction of FLS can be enhanced by pretreatment with UV or gamma-irradiation and TNF-alpha stimulation. CONCLUSION: We find that rAAV vectors have several empirical advantages for in vivo gene therapy for arthritis: (1) rAAV preferentially transduces arthritic joint cells in vivo. (2) rAAV can transduce both FLS and chondrocytes in vivo. (3) rAAV transduction of FLS can be augmented by pretreatment with agents that induce DNA repair enzymes.     

Systemic efficacy of combined suicide/cytokine gene therapy in a murine model of hepatocellular carcinoma

BACKGROUND/AIMS: Gene therapy might be a promising therapeutic approach for advanced hepatocellular carcinoma (HCC) not amenable to any effective traditional treatment. The aim of the study was to evaluate the in vitro and in vivo efficacy of combined gene therapy of HCC with two different MoMLV-derived retroviral vectors, an MFG- and a LXSN-derived vector, both containing HSV-TK and hIL-2. RESULTS: In vitro experiments on HCC cells showed efficient killing of transduced cells and efficient bystander effect after ganciclovir (GCV) treatment, with higher antitumor activity when the MFG-based vector was used. In vivo studies in a murine syngenic model of HCC demonstrated that treatment with GCV led to complete regression of tumors composed of transduced cells and regression of distant non-transduced tumors. Tumor transduction and efficacy of treatment was also demonstrated after in vivo delivery of vectors. Microarray analysis of tumor samples in mice receiving gene therapy showed up-regulation of genes involved in immune response and signal transduction. CONCLUSIONS: We demonstrated the in vitro and in vivo efficacy of combined retroviral-mediated gene therapy for HCC, with significant systemic therapeutic efficacy in vivo.     

Efficient gene transfer of CD40 ligand into primary B-CLL cells using recombinant adeno-associated virus (rAAV) vectors

B cells of chronic lymphocytic leukemia (B-CLL) are resistant to transduction with most currently available vector systems. Using an optimized adenovirus-free packaging system, recombinant adeno-associated virus (rAAV) vectors coding for the enhanced green fluorescent protein (AAV/EGFP) and CD40 ligand (AAV/CD40L) were packaged and highly purified resulting in genomic titers up to 3 x 10(11)/mL. Cells obtained from 24 patients with B-CLL were infected with AAV/EGFP or AAV/CD40L at a multiplicity of infection (MOI) of 100 resulting in transgene expression in up to 97% of cells as detected by flow cytometry 48 hours after infection. Viral transduction could be specifically blocked by heparin. Transduction with AAV/CD40L resulted in up-regulation of the costimulatory molecule CD80 not only on infected CLL cells but also on noninfected bystander leukemia B cells, whereas this effect induced specific proliferation of HLA-matched allogeneic T cells. Vaccination strategies for patients with B-CLL using leukemia cells infected ex vivo by rAAV vectors now seems possible in the near future.     

反义及小干扰RNA对大鼠肝星状细胞金属蛋白酶组织抑制因子-1表达的抑制作用

目的观察以腺相关病毒（AAV）为载体，含有针对大鼠金属蛋白酶组织抑制因子（TIMP）-1的反义RNA及小干扰RNA（siRNA）的重组AAV（rAAV／ANTI-TIMP—1／neo及rAAV／siRNA—TIMP—1／neo）感染大鼠肝星状细胞系HSC—T6后，TIMP—1表达的受抑制情况。方法经聚合酶链反应（PCR）扩增及酶切后连接，将针对TIMP—1的反义RNA片段及甄RNA片段分别构建于AAV载体中并测序鉴定，将其包装成重组病毒后感染大鼠肝星状细胞系HSC—T6，同时设空白对照组。经G418以400ug／m1的浓度筛选30d后，分别应用荧光定量PCR技术及Westernblot检测重组病毒感染组及空白对照组HSC-T6 TIMP-1的基因转录及蛋白质表达水平。结果经PCR、酶切及序列测定证实，两种重组AAV载体质粒（pd16-95／ANTI—TIMP-1／neo和pd16—95／siRNA-TIMP-1／neo）克隆成功。将重组质粒包装成病毒感染HSC—T6细胞30d后，通过荧光定量PCR技术及Westernblot分析显示，重组病毒rAAV／siRNA-TIMP-1／neo组与对照组细胞相比，HSC—T6中的TIMP-1基因的转录被抑制（P〈0．01），且表达水平与对照组相比约下降60％。而rAAV／ANTI—TIMP—1／neo组及空载体组与对照组相比TIMP—1基因的转录及表达水平差异无统计学意义（P〉0．05）。结论通过AAV载体技术重组病毒rAAV／siRNA-TIMP—1／neo可有效地抑制TIMP-1基因的表达，而针对TIMP—1基因全长的重组病毒rAAV／ANTI—TIMP—1／neo对体外培养的HSC—T6细胞TIMP—1基因的转录与表达无明显抑制作用。     

Enhancement of gene transfer with recombinant adeno-associated virus (rAAV) vectors into primary B-cell chronic lymphocytic leukemia cells by CpG-oligodeoxynucleotides

OBJECTIVE: Transduction of primary B-cell chronic lymphocytic leukemia (B-CLL) cells with recombinant adeno-associated virus (rAAV) vectors is dependent on preactivation of leukemic cells by CD40L. CpG-oligodeoxynucleotides (CpG-ODNs) are able to activate cytokine production and proliferation of B-CLL cells. Therefore CpG-ODNs were tested for their potential to enhance transgene expression in CLL cells. MATERIALS AND METHODS: Using an optimized adenovirus-free packaging system, rAAV vectors coding for the enhanced green fluorescent protein (AAV/EGFP) were packaged and highly purified resulting in infectious titers up to 5 x 10(9)/mL. Cells obtained from patients with B-CLL were infected with AAV/EGFP at a multiplicity of infection of 100 while being stimulated with CpG-ODNs and/or CD40L-expressing HeLa/SF cells. Transgene expression was assessed after 48 hours by flow cytometry. RESULTS: Stimulation of B-CLL cells by CpG-ODNs resulted in up-regulation of costimulatory molecules and G(1)/S-phase transition at similar levels compared to activation by HeLa/SF cells, but use of CpG-ODNs alone did not result in any efficient AAV/EGFP transduction. Combined stimulation of B-CLL cells with HeLa/SF cells and CpG-ODNs during AAV/EGFP transduction significantly enhanced transgene expression compared to feeder stimulation alone (p=0.004). In addition, the copy number per single cell was significantly increased by addition of CpG-ODNs as detected by quantitative real-time PCR (p=0.04). Use of self-complementary AAV vectors that are not dependent on target cell DNA synthesis did not result in increased transgene expression compared to single-stranded AAV vectors (p=0.30). CONCLUSION: Stimulation by CD40L is crucial for efficient gene transfer into B-CLL cells by rAAV vectors, whereas transduction efficiency can be significantly enhanced by CpG-ODNs.     

Recombinant adeno-associated virus serotype 9 leads to preferential cardiac transduction in vivo

Heart disease is often the end result of inherited genetic defects, which may potentially be treatable using a gene-transfer approach. Recombinant adeno-associated virus (rAAV)-mediated gene delivery has emerged as a realistic method for the treatment of such disorders. Here, we demonstrate and compare the natural affinity of specific AAV serotype capsids for transduction of cardiac tissue. We compared the previously accepted optimal rAAV serotype for transduction of skeletal muscle, rAAV2/1, with rAAV2/8 and the newer rAAV2/9 vectors carrying the CMV-lacZ construct in their respective abilities to transcend vasculature and transduce myocardium following intravenous delivery of 1x10(11) vector genomes in neonatal mice. We found that both rAAV2/8 and rAAV2/9 are able to transduce myocardium at approximately 20- and 200-fold (respectively) higher levels than rAAV2/1. Biodistribution analysis revealed that rAAV2/9 and rAAV2/8 demonstrate similar behavior in extracardiac tissue. Vector genome quantification showed an increase in genome copy numbers in cardiac tissue for several weeks following administration, which corresponds to expression data. In addition, we intravenously administered 1x10(11) vector genomes of rAAV2/9-CMV-lacZ into adult mice and achieved an expression biodistribution profile similar to that found following delivery to newborns. Although higher doses of virus will be necessary to approach those levels observed following neonatal injections, adult myocardium is also readily transduced by rAAV2/9. Finally, we have demonstrated physiological disease correction by AAV9 gene transfer in a mouse model of Pompe disease via ECG tracings and that intravenous delivery of the same vector preferentially transduces cardiac tissue in nonhuman primates.     

Herpes simplex virus thymidine kinase-mediated suicide gene therapy for hepatocellular carcinoma using HIV-1-derived lentiviral vectors

BACKGROUND/AIMS: Gene therapy is a promising approach for treatment of hepatocellular carcinoma (HCC). However, transduction of non-tumoral hepatocytes may lead to severe hepatitis when using suicide gene therapy approaches. The aim of our study was to evaluate the gene transfer efficiency into HCC cells and normal hepatocytes using human immunodeficiency virus (HIV)-derived lentiviral vectors in vitro and in vivo. METHODS: Lentiviral vectors encoding for the LacZ gene or the fusion gene HSV-Tk/GFP were tested in vitro in human HCC cells and human hepatocytes in primary culture and in vivo in a chemically induced rat model of HCC. RESULTS: We show that HIV-1-derived lentiviral vectors are efficient in transducing HCC cells in vitro and in vivo. No significant transduction of non-tumorous hepatocytes was observed in vivo whatever the route of administration used. Measurement of tumor growth following direct intratumoral injection of a lentiviral vector containing the HSV-Tk gene and GCV treatment showed a strong antitumoral efficacy in the absence of normal liver toxicity. CONCLUSIONS: These observations suggest that lentiviral vectors allow an antitumoral effect with low liver toxicity when using suicide gene therapy approach and could be efficient tools for HCC gene therapy.     

Effect of mechanical damage on ex vivo DNA virus vector-mediated gene transduction in epithelial cells of murine trachea]

The mechanism of Adenovirus (Ad) and Adeno-associated virus (AAV) vector-mediated gene transduction in murine tracheae has not been fully understood. Excised tracheae from mice were exposed to either Ad vector (Ad-CMV-LacZ) or AAV vector (AAV-CMV-LacZ) for 1 hour. LacZ gene expression in tracheal epithelial cells was detected by X-gal staining. Only patch distributions of LacZ expressing cells were observed. The percentage of LacZ expressing cells to total cells was less than 1% with either vector. Ad-mediated LacZ transduction was increased by mechanical damage using forceps. AAV-mediated gene transduction in tracheal epithelial cells was also increased by mechanical damage. Furthermore, this increased expression of vector LacZ by damaged epithelial cells was not affected by pretreatment with anti-ICAM-1 mAb or platelet-activating factor receptor antagonist. Although the Ad and AAV vectors were inefficient in transferring genetic material to murine trachea ex vivo, our results suggest that mechanical damage can enhance their transduction efficiency.     

Sex significantly influences transduction of murine liver by recombinant adeno-associated viral vectors through an androgen-dependent pathway

A systematic evaluation of the influence of sex on transduction by recombinant adeno-associated viral vector (rAAV) indicated that transgene expression after liver-targeted delivery of vector particles was between 5- to 13-fold higher in male mice compared with female mice, irrespective of the proviral promoter or cDNA and mouse strain. Molecular analysis revealed that the rAAV genome was stably retained in male liver at levels that were 7-fold higher than those observed in females. Further, the sex difference in transduction was observed with AAV-2- and AAV-5-based vectors, which use distinct receptor complexes for infection. In concordance with the differences in AAV transduction, gel shift analysis with nuclear extracts derived from the liver of mice and humans revealed substantially higher binding of host nuclear protein to the rep-binding site (RBS) of AAV inverted terminal repeat (ITR) in males compared with females. Transduction efficiency and binding of nuclear protein to RBS was dramatically reduced in male mice by castration. In contrast, although oophorectomy did not significantly influence rAAV transduction, administration of 5alpha dihydrotestosterone, prior to gene transfer, increased stable hepatocyte gene transfer in females to levels observed in male mice, implying that androgens significantly influence hepatocyte gene transfer. Interestingly, sex did not have a significant effect on AAV gene transfer into nonhepatic tissue, indicating that there are distinct tissue- and sex-specific differences in the mechanisms responsible for efficient transduction with this vector. These results have significant implications for gene therapy of autosomal and acquired disorders affecting the liver.     

Ephrin A2 receptor targeting does not increase adenoviral pancreatic cancer transduction in vivo

AIM： To generate an adenoviral vector specifically targeting the EphA2 receptor （EphA2R） highly expressed on pancreatic cancer cells in vivo. METHODS： YSA, a small peptide ligand that binds the EphA2R with high affinity, was inserted into the HI loop of the adenovirus serotype 5 fiber knob. To further increase the specificity of this vector, binding sites for native adenoviral receptors, the coxsackie and adenovirus receptor （CAR） and integrin, were ablated from the viral capsid. The ablated retargeted adenoviral vector was produced on 293T cells. Specific targeting of this novel adenoviral vector to pancreatic cancer was investigated on established human pancreatic cancer cell lines. Upon demonstrating specific in vitro targeting, in vivo targeting to subcutaneous growing human pancreatic cancer was tested by intravenous and intraperitoneal administration of the ablated adenoviral vector. RESULTS： Ablation of native cellular binding sites reduced adenoviral transduction at least 100-fold. Insertion of the YSA peptide in the HI loop restored adenoviral transduction of EphA2R-expressing cells but not of cells lacking this receptor. YSA-mediated transduction was inhibited by addition of synthetic YSA peptide. The transduction specificity of the ablated retargeted vector towards human pancreatic cancer cells was enhanced almost 10-fold in vitro. In a subsequent in vivo study in a nude （nu/nu） mouse model however, no increased adenoviral targeting to subcutaneously growing human pancreas cancer nodules was seen upon injection into the tail vein, nor upon injection into the peritoneum. CONCLUSION： Targeting the EphA2 receptor increases specificity of adenoviral transduction of human pancreatic cancer cells in vitro but fails to enhance pancreatic cancer transduction in vivo.     

Recombinant adeno-associated virus-mediated gene transfer into hematopoietic progenitor cells [published erratum appears in Blood 1995 Feb 1;85(3):862]

Recombinant adeno-associated viruses (rAAV) containing only the inverted terminal repeats (ITR) from the wild-type virus are capable of stable integration into the host cell genome, and expression of inserted genes in cultured cells. We have now defined the ability of rAAV to introduce genes into primary hematopoietic progenitors. A vector was constructed containing the coding sequences for beta- galactosidase (beta-gal), including a nuclear localization signal, under the control of a strong viral promotor. Infectious vector particles were prepared by cotransfection of the vector plasmid with a second plasmid that contained the coding sequences for AAV proteins into adenovirus-infected human embryonic kidney cells. These vector preparations transferred and expressed the beta-gal gene in human K562 erythroleukemia and Detroit 6 cells. Positive immunoselection yielded a population of enriched CD34+ cells that were transduced with the rAAV beta-gal vector. Nuclear localized enzyme expression was documented in 60% to 70% of infected cells. Progenitor-derived colonies that developed after 2 weeks in clonogenic cultures were shown to have viral- associated DNA at an estimated copy number of 1 to 2 per cell using a semiquantitative polymerase chain reaction (PCR) method. Integration of AAV into hematopoietic progenitors was documented using wild-type virus, as its genome may integrate at a preferred site on chromosome 19. Our data suggest that rAAV will transfer and express genes in primitive hematopoietic progenitors with high frequency, and support the development of this vector system for therapeutic gene transfer.     

Recombinant AAV2 transduction of primitive human hematopoietic stem cells capable of serial engraftment in immune-deficient mice

A recombinant AAV2 (rAAV2) vector encoding antisense RNA to HIV-1 transactivating region (TAR) was evaluated for transduction of human cord blood CD34+CD38- hematopoietic stem cells (HSC) capable of serial engraftment in nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. Results revealed long-term multilineage marking in primary and secondary recipients, and significantly, an enrichment of transduced cells in secondary hosts, indicating efficient transduction of multipotential self-renewing HSC. These results were confirmed by the persistence of rAAV marking of clonogenic progenitors in serial analyses of recipient marrow. Upon HIV-1 challenge, the macrophage progeny of transduced CD34+ cells expressed antisense RNA and exhibited sustained and significant inhibition of virus replication as compared with controls in every donor tested, without selective pressure. This study represents a clear in vivo demonstration of efficient rAAV2 transduction of human HSC.     

Enhanced gene transfer to arthritic joints using adeno-associated virus type 5: implications for intra-articular gene therapy

BACKGROUND: Gene therapy of the joint has great potential as a new therapeutic approach for the treatment of rheumatoid arthritis (RA). The vector chosen is of crucial importance for clinical success. OBJECTIVE: To investigate the tropism and transduction efficiency in arthritic joints in vivo, and in synovial cells in vitro, using five different serotypes of recombinant adeno-associated virus (rAAV) encoding beta-galactosidase or green fluorescent protein genes. METHODS: rAAV was injected into the ankle joints of rats with adjuvant arthritis after the onset of disease. Synovial tissue was examined at different time points for beta-galactosidase protein and gene expression by in situ staining and polymerase chain reaction (PCR) analysis, respectively. In addition, the ability of rAAV to transduce primary human fibroblast-like synoviocytes from patients with RA was investigated in vitro. RESULTS: Intra-articular injection of the rAAV5 serotype resulted in the highest synovial transduction, followed by much lower expression using rAAV2. Expression of the transgene was already detectable 7 days after injection and lasted for at least 4 weeks. Only background staining was seen for serotypes 1, 3, and 4. Importantly, there was a minimal humoral immune response to rAAV5 compared with rAAV2. Additionally, it was found that both rAAV2 and rAAV5 can efficiently transduce human fibroblast-like synoviocytes obtained from patients with RA. CONCLUSION: Intra-articular rAAV mediated gene therapy in RA might be improved by using rAAV5 rather than other serotypes.     

Variables pertinent to the efficiency of adeno-associated virus (AAV) vectors mediated gene transfer to human vascular endothelial cells.

Factors influencing adeno-associated virus (AAV) - mediated gene transfer to endothelial cells are not fully determined. We tested the variables pertinent to the efficiency of AAV-mediated gene transfer to human vascular endothelial cells (HUVEC) including: (i) kinetics of transduction efficiency of LacZ gene to HUVEC, (ii) the concentration and volume of vector-containing medium, (iii) the period of incubation time of AAV vectors with HUVEC, (iv) the target cell density/proliferation, (v) the duration of transgene expression. There is a dose-response relationship between moi of vectors and transduction efficiency in HUVEC. The higher moi of AAV vectors achieved more than 80% of transduction efficiency in cultured HUVEC. AAV vectors showed incubation time dependent increase in transduction efficiency of LacZ gene to the HUVEC up to 24 h of vector exposure. The foreign gene of AAV vectors preferably transduces the lower density of cells being proliferated. These results indicate that AAV-vector is efficient for gene transfer to HUVEC, and higher moi of vectors or a longer period exposure of vectors to proliferating HUVEC can facilitate efficient tranduction of foreign gene into human vascular endothelial cells in vitro.     

Adenovirus-mediated CD40 ligand gene therapy in a rat model of orthotopic hepatocellular carcinoma.

Unresectable hepatocellular carcinoma (HCC) lacks effective therapy and entails very poor prognosis. In this study, we have explored a gene-therapeutic approach to stimulate antitumor immunity by adenoviral-mediated transfer of CD40 ligand to treat HCC in rats. In vitro infection of a rat HCC cell line (McA-RH7777) with adenoviral vector expressing CD40 ligand (AdCMVmCD40L) induced CD40L expression in a dose-dependent manner. Expression of CD40L in McA-RH7777 cells did not alter their growth rate in vitro, but it abrogated their tumorigenicity when CD40L-expressing cells were implanted into the liver of syngenic Buffalo rats. In vivo gene therapy of established orthotopic HCC nodules (6.5 mm in diameter) in Buffalo rats by intratumor injection of AdCMVmCD40L vector led to complete tumor eradication and long-term survival in 69.5% of treated animals. Therapy with AdCMVmCD40L induced strong lymphocytic infiltration of the tumoral tissue and increased apoptosis of malignant cells. The observed antitumoral effect was mediated by CD8(+) T cells and was associated with increased interleukin (IL)-12 serum levels and enhanced natural killer (NK) activity. Animals that eliminated the tumor after in vivo gene therapy developed protective antitumor immunity being resistant to rechallenge with neoplastic cells. Toxicity of the therapy with AdCMVmCD40L was slight, with only a transient increase in the level of serum transaminases and minor lymphocyte infiltration of normal liver tissue. These data demonstrate that intratumoral administration of AdCMVmCD40L may provide an efficient and safe treatment for HCC.     

Recombinant adeno-associated virus preferentially transduces human,compared to mouse,synovium: implications for arthritis therapy

Abstract

Despite a number of published reports, including from our own laboratory, suggesting that adeno-associated virus (AAV) transduces mouse synovium, a careful analysis demonstrated transduction predominantly of the subsynovial muscle tissue, while the synovial lining is poorly transduced. To investigate the potential of AAV to transduce human synovium, three human rheumatoid arthritis (RA) and two murine collagen-induced arthritis (CIA) synovial cell lines were infected with recombinant AAV (rAAV) vectors encoding either mouse IL-10 or IL-4. Low-level transgene expression was observed. However, either Γ-irradiation or the addition of a low-titer E1-, E3-deleted recombinant adenovirus resulted in up to a 100-fold increase in transgene product in the human, but not the mouse, cell lines. RA synovial tissues implanted subcutaneously in severe combined immunodeficiency (SCID) mice, which were subsequently infected with rAAV, showed marked increases in transgene expression when co-infected with adenovirus. To our knowledge, this is the first study to show that intact human synovial tissues can be transduced by rAAV, and it suggests that murine arthritis may not be an optimal model to study rAAV as a gene transfer vector. Further studies to elucidate the mechanisms limiting gene transduction in human synovium may allow optimization of this vector for the treatment of arthritis.     

Gene therapy for colon cancer by adeno-associated viral vector-mediated transfer of survivin Cys84Ala mutant

BACKGROUND AND AIMS: Reactivation of survivin expression is involved in carcinogenesis and angiogenesis in colon cancer. Previous in vitro studies showed that mutation of the cysteine residue at position 84 (Cys84Ala) of survivin generates a dominant-negative mutant that triggers mitotic catastrophe and apoptosis. We investigated the therapeutic effect of the adeno-associated virus (AAV)-mediated survivin mutant (Cys84Ala) on colon cancer. METHODS: Survivin mutant (Cys84Ala) (Sur-Mut(Cys84Ala)) was cloned into the AAV expression vector pAM/CAG-WPRE.poly(A) to generate recombinant AAV-Sur-Mut(Cys84Ala) virus. Cell proliferation, apoptosis, mitotic catastrophe, and tumor growth were measured in vitro and in vivo. RESULTS: Transduction of colon cancer cells with rAAV-Sur-Mut(Cys84Ala) inhibited cell proliferation and induced apoptosis and mitotic catastrophe in vitro. rAAV-Sur-Mut(Cys84Ala) sensitized colon cancer cells to chemotherapeutic drugs. Furthermore, expression of survivin mutant mediated by AAV inhibited tumorigenesis in colon cancer cells. Intratumoral injection of rAAV-Sur-Mut(Cys84Ala) significantly induced apoptosis and mitotic catastrophe and inhibited angiogenesis and tumor growth in a colon cancer xenograft model in vivo. No obvious cytotoxicity to other tissues was observed. More importantly, rAAV-Sur-Mut(Cys84Ala) expression strongly enhanced the antitumor activity of 5-Fluorouracil (5-FU), resulting in regression of established tumors. CONCLUSIONS: Our results showed that rAAV-Sur-Mut(Cys84Ala) induced apoptosis and mitotic catastrophe and inhibited tumor angiogenesis and tumor growth. Thus, use of AAV-mediated survivin mutant (Cys84Ala) is a promising strategy in colon cancer gene therapy.     

Adeno-associated virus mediated endostatin gene therapy in combination with topoisomerase inhibitor effectively controls liver tumor in mouse model

AIM: rAAV mediated endostatin gene therapy has been examined as a new method for treating cancer. However, a sustained and high protein delivery is required to achieve the desired therapeutic effects. We evaluated the impact of topoisomerase inhibitors in rAAV delivered endostatin gene therapy in a liver tumor model. METHODS: rAAV containing endostatin expression cassettes were transduced into hepatoma cell lines. To test whether the topoisomerase inhibitor pretreatment increased the expression of endostatin, Western blotting and ELISA were performed. The biologic activity of endostatin was confirmed by endothelial cell proliferation and tube formation assays. The anti-tumor effects of the rAAV-endostatin vector combined with a topoisomerase inhibitor, etoposide, were evaluated in a mouse liver tumor model. RESULTS: Topoisomerase inhibitors, including camptothecin and etoposide, were found to increase the endostatin expression level in vitro. The over-expressed endostatin, as a result of pretreatment with a topoisomerase inhibitor, was also biologically active. In animal experiments, the combined therapy of topoisomerase inhibitor, etoposide with the rAAV-endostatin vector had the best tumor-suppressive effect and tumor foci were barely observed in livers of the treated mice. Pretreatment with an etoposide increased the level of endostatin in the liver and serum of rAAV-endostatin treated mice. Finally, the mice treated with rAAV-endostatin in combination with etoposide showed the longest survival among the experimental models. CONCLUSION: rAAV delivered endostatin gene therapy in combination with a topoisomerase inhibitor pretreatment is an effective modality for anticancer gene therapy.     

Gene transfer and expression of enhanced green fluorescent protein in variant HT-29c cells

AIM: To study the expression of enhanced green fluorescent protein (EGFP) gene in retrovirally transduced variant HT-29 cells. METHODS: The retroviral vector prkat EGFP/neo was constructed and transfected into the 293T cell using a standard calcium phosphate precipitation method. HT-29c cells (selected from HT-29 cells) were transduced by a retroviral vector encoding the GEFP gene. The fluorescence intensity of colorectal carcinoma HT-29c cells after transduced with the EGFP bearing retrovirus was visualized using fluorescence microscope and fluorescence activated cell sorter (FACS) analysis. Multiple biological behaviors of transduced cells such as the proliferating potential and the expression of various antigens were comparatively analyzed between untransduced and transduced cells in vitro. EGFP expression of the fresh tumor tissue was assessed in vivo. RESULTS: After transduced, HT-29c cells displayed a stable and long-term EGFP expression under the nonselective conditions in vitro. After cells were successively cultured to passage 50 in vitro, EGFP expression was still at a high level. Their biological behaviors, such as expression of tumor antigens, proliferation rate and aggregation capability were not different compared to untransduced parental cells in vitro. In subcutaneous tumors, EGFP was stable and highly expressed. CONCLUSION: An EGFP expressing retroviral vector was used to transduce HT-29c cells. The transduced cells show a stable and long-term EGFP expression in vitro and in vivo. These cells with EGFP are a valuable tool for in vivo research of tumor metastatic spread.