Tool For Cancer Gene Therapy Research Articles

A Replication-Competent Retroviral Vector Expressing the HERV-W Envelope Glycoprotein is a Potential Tool for Cancer Gene Therapy.

The fusogenic membrane glycoprotein (FMG) derived from the human endogenous retrovirus-W (HERV-W) exhibits fusogenic properties, making it a promising candidate for cancer gene therapy. When cells are transfected with HERV-W FMG, they can fuse with neighboring cells expressing the receptor, resulting in the formation of syncytia. These syncytia eventually undergo cell death within a few days. In addition, it has been observed that an HERV-W env mutant, which is truncated after amino acid 483, displays increased fusogenicity compared to the wild-type HERV-W env. In this study, we observed syncytium formation upon transfection of HeLa and TE671 human cancer cells with plasmids containing the HERV-W 483 gene. To explore the potential of a semi-replication-competent retroviral (s-RCR) vector encoding HERV-W 483 for FMG-mediated cancer gene therapy, we developed two replication-defective retroviral vectors: a gag-pol vector encoding HERV-W 483 (MoMLV-HERV-W 483) and an env vector encoding VSV-G (pCLXSN-VSV-G-EGFP). When MoMLV-HERV-W 483 and pCLXSN-VSV-G-EGFP were co-transfected into HEK293T cells to produce the s-RCR vector, gradual syncytium formation was observed. However, the titers of the s-RCR virus remained consistently low. To enhance gene transfer efficiency, we constructed an RCR vector encoding HERV-W 483 (MoMLV-10A1-HERV-W 483), which demonstrated replication ability in HEK293T cells. Infection of A549 and HT1080 human cancer cell lines with this RCR vector induced syncytium formation and subsequent cell death. Consequently, both the s-RCR vector and RCR encoding HERV-W 483 hold promise as valuable tools for cancer gene therapy.

Hierarchically tumor-activated nanoCRISPR-Cas13a facilitates efficient microRNA disruption for multi-pathway-mediated tumor suppression.

Rationale: CRISPR-Cas13a is an efficient tool for robust RNA knockdown with lower off-target effect, which may be a potentially powerful and safe tool for cancer gene therapy. However, therapeutic effect of current cancer gene therapy that targeting monogene was compromised by the multi-mutational signal pathway alterations of tumorigenesis. Methods: Here, hierarchically tumor-activated nanoCRISPR-Cas13a (CHAIN) is fabricated for multi-pathway-mediated tumor suppression by efficient microRNA disruption in vivo. A fluorinated polyetherimide (PEI; Mw=1.8KD) with graft rate of 33% (PF33) was utilized to compact the CRISPR-Cas13a megaplasmid targeting microRNA-21 (miR-21) (pCas13a-crRNA) via self-assemble to constitute a nanoscale 'core' (PF33/pCas13a-crRNA), which was further wrapped by modified hyaluronan (HA) derivatives (galactopyranoside-PEG2000-HA, GPH) to form CHAIN. Results: The dual-tumor-targeting and tumor-activated CHAIN not only manifested long-term circulation, but augmented tumor cellular uptake and endo/lysosomal escape, thus achieving efficient transfection of CRISPR-Cas13a megaplasmid (~ 13 kb) in tumor cells with minimal toxity. Efficient knockdown of miR-21 by CHAIN restored programmed cell death protein 4 (PDCD4) and reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) and further crippled downstream matrix metalloproteinases-2 (MMP-2), which undermined cancer proliferation, migration and invasion. Meanwhile, the miR-21-PDCD4-AP-1 positive feedback loop further functioned as an enhanced force for anti-tumor activity. Conclusion: Treatment with CHAIN in hepatocellular carcinoma mouse model achieved significant inhibition of miR-21 expression and rescued multi-pathway, which triggered substantial tumor growth suppression. By efficient CRISPR-Cas13a induced interference of one oncogenic microRNA, the CHAIN platform exerted promising capabilities in cancer treatment.

Dual-RNA controlled delivery system inhibited tumor growth by apoptosis induction and TME activation

Drug-controlled release is recognized as effective for improving compliance with treatment and obtaining better therapeutic efficacy with less toxicity in cancer treatment. However, few reports in this area are involved in nucleic acids delivery, especially in RNA therapeutics delivery. In this study, an injectable hydrogel Methacrylated gelatin (GM) scaffold was introduced into a dual-RNA hybrid delivery complex hybrid lipid particle (HLP) to form a G-HLP/RNAs system. This system can control the release of both siRNA and mRNA and was found to be efficient for protecting these RNAs from biodegradation and retaining their therapeutic effect over 7 days. Further, a tumor environment (TME)-activation function after peritumoral injection of mocked GM scaffold was observed. Then, matured DC cells and activated T-cells were detected by the addition of HLP/RNAs complex, thus verifying the immunoactivation function of GM scaffold and its ability to reserve immune cells and antigens. Finally, two doses of G-HLP/RNAs treatment efficiently suppressed C26 tumor growth in mice with a tumor weight inhibition rate of 71.9%. Owing to its ability to achieve RNA drug-controlled release, alter TME, and induce tumor apoptosis, the G-HLP/RNAs system may become a valuable tool for cancer gene therapy.

CRISPR-to-Kill (C2K)-Employing the Bacterial Immune System to Kill Cancer Cells.

Simple SummaryReasoning that multiple DNA breaks will trigger programmed cell death, we generated lentiviral CRISPR-to-kill (C2K) vectors targeting highly repetitive SINE sequences for cancer gene therapy. In proof-of-concept experiments, C2K-Alu-vectors selectively killed human, but not murine cell lines, and efficiently inhibited the growth of patient-derived glioblastoma cell lines resistant to high-dose irradiation. In combination with tumor-targeting approaches, the C2K system might represent a promising tool for cancer gene therapy.CRISPR/Cas9 was described as a bacterial immune system that uses targeted introduction of DNA double-strand breaks (DSBs) to destroy invaders. We hypothesized that we can analogously employ CRISPR/Cas9 nucleases to kill cancer cells by inducing maximal numbers of DSBs in their genome and thus triggering programmed cell death. To do so, we generated CRISPR-to-kill (C2K) lentiviral particles targeting highly repetitive Short Interspersed Nuclear Element-Alu sequences. Our Alu-specific sgRNA has more than 15,000 perfectly matched target sites within the human genome. C2K-Alu-vectors selectively killed human, but not murine cell lines. More importantly, they efficiently inhibited the growth of cancer cells including patient-derived glioblastoma cell lines resistant to high-dose irradiation. Our data provide proof-of-concept for the potential of C2K as a novel treatment strategy overcoming common resistance mechanisms. In combination with tumor-targeting approaches, the C2K system might therefore represent a promising tool for cancer gene therapy.

Mesenchymal Stem Cells Engineered by Nonviral Vectors: A Powerful Tool in Cancer Gene Therapy.

Due to their “tumor homing” and “immune privilege” characteristics, the use of mesenchymal stem cells (MSCs) has been proposed as a novel tool against cancer. MSCs are genetically engineered in vitro and then utilized to deliver tumoricidal agents, including prodrugs and bioactive molecules, to tumors. The genetic modification of MSCs can be achieved by various vectors, and in most cases viral vectors are used; however, viruses may be associated with carcinogenesis and immunogenicity, restricting their clinical translational potential. As such, nonviral vectors have emerged as a potential solution to address these limitations and have gradually attracted increasing attention. In this review, we briefly revisit the current knowledge about MSC-based cancer gene therapy. Then, we summarize the advantages and challenges of nonviral vectors for MSC transfection. Finally, we discuss recent advances in the development of new nonviral vectors, which have provided promising strategies to overcome obstacles in the gene modulation of MSCs.

N-Isopropylacrylamide-modified polyethylenimine-mediated miR-29a delivery to inhibit the proliferation and migration of lung cancer cells

MicroRNAs have been identified as a promising tool in cancer gene therapy, and an efficient and safe gene carrier was significantly required in the clinical application of miRNAs. Herein, a polyethylenimine (PEI) derivative, N-isopropylacrylamide-modified PEI (namely PEN), was constructed through Michael addition and then employed as a carrier for miR-29a transfection. The carrier PEN has been demonstrated to possess favorable ability to condense miR-29a into stable nanoparticles and protect miR-29a against the nuclease degradation, using agarose gel retardation assay. Meanwhile, PEN exhibited excellent efficiency in miR-29a transfection demonstrated by flow cytometry and confocal laser scanning microscope. Further, the PEN-mediated miR-29a transfection could achieve an obvious anti-proliferative effect owing to the activation of cell apoptosis and the cell cycle arrest at G1 phase, using human lung adenocarcinoma cell line A549 as a model. In addition, PEN/miR-29a nanoparticles could suppress the migration and invasion of cancer cells measured by wound healing and Transwell migration assays. Overall, the PEN-mediated miR-29a transfection could be potentially employed as a useful approach to achieve cancer gene therapy.

Lauroylated Histidine-Enriched S413-PV Peptide as an Efficient Gene Silencing Mediator in Cancer Cells.

This study aimed to endow the cell-penetrating peptide (CPP) S413-PV with adequate features towards a safe and effective application in cancer gene therapy. Peptide/siRNA complexes were prepared with two new derivatives of the CPP S413-PV, which combine a lauroyl group attached to the N- or C-terminus with a histidine-enrichment in the N-terminus of the S413-PV peptide, being named C12-H5-S413-PV and H5-S413-PV-C12, respectively. Physicochemical characterization of siRNA complexes was performed and their cytotoxicity and efficiency to mediate siRNA delivery and gene silencing in cancer cells were assessed in the absence and presence of serum. Peptide/siRNA complexes prepared with the C12-H5-S413-PV derivative showed a nanoscale (ca. 100nm) particle size, as revealed by TEM, and efficiently mediated gene silencing (37%) in human U87 glioblastoma cells in the presence of 30% serum. In addition, the new C12-H5-S413-PV-based siRNA delivery system efficiently downregulated stearoyl-CoA desaturase-1, a key-enzyme of lipid metabolism overexpressed in cancer, which resulted in a significant decrease in the viability of U87 cells. Importantly, these complexes were able to spare healthy human astrocytes. These encouraging results pave the way for a potential application of the C12-H5-S413-PV peptide as a promising tool in cancer gene therapy.

Construction of a replication-competent retroviral vector for expression of the VSV-G envelope glycoprotein for cancer gene therapy.

Gibbon ape leukemia virus (GALV) can infect a wide variety of cells but fails to infect most cells derived from laboratory mice. Transduction of human hematopoietic stem cells with GALV retroviral vectors is more efficient than with amphotropic vectors. In this study, a Moloney murine leukemia virus-gibbon ape leukemia virus (MoMLV-GALV) vector was constructed by replacing the natural env gene of the full-length Moloney MLV genome with the GALV env gene. To monitor viral transmission by green fluorescent protein (GFP) expression, internal ribosomal entry site-enhanced GFP (IRES-EGFP) was positioned between the GALV env gene and the 3' untranslated region (3' UTR) to obtain pMoMLV-GALV-EGFP. The MoMLV-GALV-EGFP vector was able to replicate with high titer in TE671 human rhabdomyosarcoma cells and U-87 human glioma cells. To evaluate the potential of the MoMLV-GALV vector as a therapeutic agent, the gene for the fusogenic envelope G glycoprotein of vesicular stomatitis virus (VSV-G) was incorporated into the vector. Infection with the resulting MoMLV-GALV-VSV-G vector resulted in lysis of the U-87 cells due to syncytium formation. Syncytium formation was also observed in the transfected human prostate cancer cell line LNCaP after extended cultivation of cells. In addition, we deleted the GALV env gene from the MoMLV-GALV-VSV-G vector to improve viral genome stability. This MoMLV-VSV-G vector is also replication competent and induces syncytium formation in 293T, HT1080, TE671 and U-87 cells. These results suggest that replication of the MoMLV-GALV-VSV-G vector or MoMLV-VSV-G vector may directly lead to cytotoxicity. Therefore, the vectors developed in this study are potentially useful tools for cancer gene therapy.

LdrB Toxin with In Vitro and In Vivo Antitumor Activity as a Potential Tool for Cancer Gene Therapy.

Due to the high prevalence of cancer in recent years, it is necessary to develop new and more effective therapies that produce fewer side effects. Development of gene therapy for cancer based on the use of suicide genes that can damage the tumor cell, without requiring a prodrug for its lethal effect, is one of the recent foci of gene therapy strategies. We evaluated the cytotoxic impact of the LdrB toxin from Escherichia coli k12 as a possible tool for cancer gene therapy. For that, colorectal and breast cancer cells were transfected under the control of a TRE3G promoter inducible by doxycycline. Our results showed that ldrB gene expression induced a drastic inhibition of proliferation in vitro, in both 2D and 3D experimental models. Moreover, unlike conventional chemotherapy, the ldrB gene induced a severe loss of proliferation in vivo without any side effects in our animal model. This antitumor outcome was modulated by cell cycle arrest in the G0/G1 phase and apoptotic death. Scanning electronic microscopy demonstrates that the LdrB toxin conserves its pore-forming ability in HCT-116 cells as in E. coli k12. Taken together, our results provide, for the first time, a proof of concept of the antitumor capacity of the ldrB gene in colorectal and breast cancer.

Interferon-Inducible Mx Promoter-Driven, Long-Term Transgene Expression System of Interferon-β for Cancer Gene Therapy.

Gene therapy techniques aiming to induce the long-term interferon-β (IFN-β) expression are desirable for inhibiting cancer growth. However, there has been no success in this regard because IFN-β significantly inhibits transgene expression. This study used the IFN-inducible Mx promoter to promote IFN-β expression. The pMx-IFN-β plasmid was constructed to achieve long-term IFN-β expression. In cultured cells transfected with the Mx promoter-driven reporter protein plasmid, IFN-β induced concentration-dependent expression of the reporter protein. After the hydrodynamic injection of pMx-IFN-β into mice, the serum concentration of IFN-β was maintained at ≥100 pg/mL for >1 month. IFN-β expression was significantly suppressed by the co-injection of small interfering RNA targeting the interferon-α/β receptor (IFNAR), suggesting that IFN-β binding to IFNAR increased IFN-β expression. Moreover, the hydrodynamic injection of pMx-IFN-β significantly suppressed the growth of colon26 tumors in mice. In contrast, a conventional promoter-driven plasmid was less effective than pMx-IFN-β in all the experiments. Taken together, these results indicate that the interferon-inducible Mx promoter-driven expression system effectively achieves long-term expression of IFN-β and represents a potential tool for cancer gene therapy.

Adenoviral Vector Designed for p53 Dependent Transgene Expression and Replication as a Potential Tool for Cancer Gene Therapy

Background: A major obstacle of adenoviral gene therapy of cancer is the restriction of therapeutic gene expression or viral replication to malignant cells to avoid severe side effects on healthy tissue. In this study we present the bicistronic adenoviral vector Adp53dpR for gene therapy of cancer cells with altered p53-status.

Genetically Modified Murine Adipose-Derived Mesenchymal Stem Cells Producing Interleukin-2 Favor B16F10 Melanoma Cell Proliferation

Adipose-derived mesenchymal stem cells (ADSCs) are attractive tools for cancer gene therapy due to their intrinsic tropism to the tumor environment. Interleukin-2 (IL2) is recognized as a key regulatory molecule, which enhances the activity and growth of the immune effector cell function. High-Dose IL2 Therapy is an option for treatment of malignant melanoma but has frequent, often serious and sometimes life-threatening side effects. Here we investigated the effect of genetically modified ADSCs (GM-ADSCs) expressing IL2 in immunocompetent mouse models of subcutaneous and lung metastatic melanoma. Prior to in vivo studies, we demonstrated that IL2 produced by GM-ADSCs may act as a growth factor for melanoma cells due to the increased viability and reduced apoptosis of melanoma cells after in vitro treatment. Subcutaneous co-injection of IL2-expressing ADSCs with melanoma cells significantly enhanced the melanoma tumor growth. Furthermore, histological analysis of subcutaneous tumors for IL2 and Melan-A (a melanocytic differentiation marker) confirmed that most of cells in melanoma/IL2-ADSC co-injected tumors are melanoma cells, not IL2-ADSCs. In pulmonary metastases model, melanoma cells were injected intravenously and 10 days later mice were treated by systematical injection of GM-ADSCs. Intravenously injected IL2-ADSCs engrafted into melanoma lung tumors but were unable to reduce melanoma lung metastases. Besides, administered IL2-ADSCs significantly reduced systemic CD4+ cells and did not impact the total survival of lung metastases melanoma bearing mice. In conclusion, this study showed that IL2-producing ADSCs can favor B16F10 melanoma cell proliferation. Therefore, therapies utilizing IL2 have to be taken into careful consideration.

Safety profiles and antitumor efficacy of oncolytic adenovirus coated with bioreducible polymer in the treatment of a CAR negative tumor model.

Adenovirus (Ad) vectors show promise as cancer gene therapy delivery vehicles, but immunogenic safety concerns and coxsackie and adenovirus receptor (CAR)-dependency have limited their use. Alternately, biocompatible and bioreducible nonviral vectors, including arginine-grafted cationic polymers, have been shown to deliver nucleic acids through a cell penetration peptide (CPP) and protein transduction domain (PTD) effect. We utilized the advantages of both viral and nonviral vectors to develop a hybrid gene delivery vehicle by coating Ad with mPEG-PEI-g-Arg-S-S-Arg-g-PEI-mPEG (Ad/PPSA). Characterization of Ad/PPSA particle size and zeta potential showed an overall size and cationic charge increase in a polymer concentration-dependent manner. Ad/PPSA also showed a marked transduction efficiency increase in both CAR-negative and -positive cells compared to naked Ad. Competition assays demonstrated that Ad/PPSA produced higher transgene expression levels than naked Ad and achieved CAR-independent transduction. Oncolytic Ad (DWP418)/PPSA was able to overcome the nonspecificity of polymer-only therapies by demonstrating cancer-specific killing effects. Furthermore, the DWP418/PPSA nanocomplex elicited a 2.24-fold greater antitumor efficacy than naked Ad in vivo. This was supported by immunohistochemical confirmation of Ad E1As accumulation in MCF7 xenografted tumors. Lastly, intravenous injection of DWP418/PPSA elicited less innate immune response compared to naked Ad, evaluated by interleukin-6 cytokine release into the serum. The increased antitumor effect and improved vector targeting to both CAR-negative and -positive cells make DWP418/PPSA a promising tool for cancer gene therapy.

Application of Thermoresponsive PNIPAAM-b-PAMPTMA Diblock Copolymers in siRNA Delivery

Gene knockdown has emerged as an important tool for cancer gene therapy as well as for viral infections and dominantly inherited genetic disorders. The generation of suitable siRNA delivery systems poses some challenges, namely, to avoid nuclease degradation, to surpass the cytoplasmic membrane, and to release the nucleic acids into the cytosol. Aiming at evaluating the ability of thermoresponsive block copolymers formed by units of N-isopropylacrylamide and of (3-acrylamidopropyl)trimethylammonium chloride to efficiently deliver siRNAs, an extensive study was performed with four different copolymers using a human fibrosarcoma cell line as cell model. The silencing ability and cytotoxicity of the generated copolymer-based siRNA delivery systems were found to be dependent on the cloud point of the polymer, which corresponds to the transition temperature at which the aggregation or precipitation of the polymer molecules becomes thermodynamically more favorable than their solubilization. In the present study, a system capable of delivering siRNAs efficiently, specifically and without presenting relevant cytotoxicity, even in the presence of serum, was developed. Confocal fluorescence experiments showed that the ability of the generated systems to silence the target gene is related to some extent to nucleic acid internalization, being also dependent on polymer/siRNA dissociation at 37 °C. Thus, a delicate balance between nucleic acid internalization and intracellular release must be met in order to reach an ideal knockdown efficiency. The special features and potential for manipulation of the N-isopropylacrylamide-based copolymers make them suitable materials for the design and synthesis of new and promising siRNA delivery systems.

The dual effect of ultrasound-targeted microbubble destruction in mediating recombinant adeno-associated virus delivery in renal cell carcinoma: transfection enhancement and tumor inhibition

Recombinant adeno-associated virus (rAAV) is recognized as a promising vector for cancer gene therapy, although its low transfer efficiency in less permissive cells limits extensive application. Our previous studies reported that ultrasound-targeted microbubble (MB) destruction (UTMD) enhanced rAAV transfer in its permissive retinal cells. In the present study, we investigated whether UTMD increased rAAV transfer in less permissive human renal cell carcinoma (hRCC) cells and tumors. hRCC cells were treated with rAAV2 under different conditions of UTMD, and the viral transfer efficiency and cell viability were analyzed. Fifty-two male nude mice (BALB/c) implanted with hRCC cells were randomly assigned to four groups consisting of rAAV, rAAV + ultrasound and rAAV + UTMD (20 µl and 40 µl of MBs). UTMD was initiated immediately after intratumoral viral injection, and viral transfer efficiency and tumor volumes were analyzed at 12 weeks after infection. The efficiency of non-augmented transfer of rAAV2 into hRCC cells was low (17.28 ± 2.44%). The use of UTMD enhanced viral transfer efficiency by two- to three-fold, and enhanced viral genomic DNA by more than nine-fold, without decreasing cell viability. In vivo studies also showed that UTMD increased rAAV2 transfer in tumor. The enhancements were maintained for a period of 12 weeks. Tumor growth in mice was inhibited by UTMD treatment, and UTMD treatment augmented by MBs (40 µl) produced an even stronger effect. UTMD enhanced rAAV2 transfer into less permissive RCC cells and tumors, resulting in inhibition of tumor growth, which suggests that UTMD may be a useful delivery tool for cancer gene therapy.

Aptamer modification improves the adenoviral transduction of malignant glioma cells

Adenovirus has shown increasing promise in the gene-viral therapy for glioblastoma, a treatment strategy that relies on the delivery of viruses or transgenes into tumor cells. However, targeting of adenovirus to human glioblastoma remains a challenge due to the low expression level of coxsackie and adenovirus receptor (CAR) in glioma cells. Aptamers are small and highly structured single-stranded oligonucleotides that bind at high affinity to a target molecule, and are good candidates for targeted imaging and therapy. In this study, to construct an aptamer-modified Ad5, we first genetically modified the HVR5 of Ad hexon by biotin acceptor peptide (BAP), which would be metabolically biotinylated during production in HEK293 cells, and then attached the biotin labeled aptamer to the modified Ad through avidin–biotin binding. The aptamers used in this study includes AS1411 and GBI-10. The former is a DNA aptamer that can bind to nucleolin, a nuclear matrix protein found on the surface of cancer cells. The latter is a DNA aptamer that can recognize the extracellular matrix protein tenascin-C on the surface of human glioblastoma cells. To examine if aptamer-modification of the hexon protein could improve the adenoviral transduction efficiency, a glioblastoma cell line, U251, was transduced with aptamer-modified Ads. The transduction efficiency of AS1411- or GBI-10-modified Ad was approximately 4.1-fold or 5.2-fold higher than that of the control. The data indicated that aptamer modified adenovirus would be a useful tool for cancer gene therapy.

Polypurine reverse Hoogsteen hairpins as a gene therapy tool against survivin in human prostate cancer PC3 cells in vitro and in vivo

As a new approach for gene therapy, we recently developed a new type of molecule called polypurine reverse Hoogsteen hairpins (PPRHs). We decided to explore the in vitro and in vivo effect of PPRHs in cancer choosing survivin as a target since it is involved in apoptosis, mitosis and angiogenesis, and overexpressed in different tumors. We designed four PPRHs against the survivin gene, one of them directed against the template strand and three against different regions of the coding strand. These PPRHs were tested in PC3 prostate cancer cells in an in vitro screening of cell viability and apoptosis. PPRHs against the promoter sequence were the most effective and caused a decrease in survivin mRNA and protein levels. We confirmed the binding between the selected PPRHs and their target sequences in the survivin gene. In addition we determined that both the template- and the coding-PPRH targeting the survivin promoter were interfering with the binding of transcription factors Sp1 and GATA-3, respectively. Finally, we conducted two in vivo efficacy assays using the Coding-PPRH against the survivin promoter and performing two routes of administration, namely intratumoral and intravenous, in a subcutaneous xenograft tumor model of PC3 prostate cancer cells. The results showed that the chosen Coding-PPRH proved to be effective in decreasing tumor volume, and reduced the levels of survivin protein and the formation of blood vessels. These findings represent the preclinical proof of principle of PPRHs as a new silencing tool for cancer gene therapy.

An Epidermal Growth Factor Motif from Del1 Protein Increases the Efficiency of In Vivo Gene Transfer with a Non-Viral Vector

Increasing the efficiency of gene transfer using non-viral vectors, which have the potential to be safe and economical, would improve upon available options for gene therapy. We previously reported that the third EGF motif of the extracellular matrix protein Del1 (E3) increases the transfection efficiency of non-viral vector methods. Here, we asked if E3 could increase the in vivo transfection efficiency of a polyplex-based approach. To test this, cDNA encoding a heat-stable alkaline phosphatase (AP) was first injected intravenously into mice along with recombinant E3. After 24h, exogenous AP activity in serum was measured. We found that the introduction of E3 resulted in 50% more AP activity as compared to the control. We next tested transfection into a tumour explant of SCCKN cells, an oral carcinoma-derived cell line. To do this, a cDNA encoding yellow fluorescent protein was locally injected into a tumour explant, followed by local injection of recombinant E3. Use of E3 increased the number of transfected cells to 2.5 times that of the control. Histochemical staining revealed that E3-induced apoptosis in a tumour explant. The data suggest that E3 might be a useful tool for cancer gene therapy using non-viral vectors.

Semliki Forest virus biodistribution in tumor-free and 4T1 mammary tumor-bearing mice: a comparison of transgene delivery by recombinant virus particles and naked RNA replicon

Semliki Forest virus (SFV) vectors are promising tools for cancer gene therapy because they ensure a high level of transgene expression and a rapid and strong cytopathic effect. However, broad tissue tropism and transient expression make it more difficult to develop an optimal cancer treatment strategy. In this study, we have compared the distribution of recombinant SFV particles (recSFV) and naked viral RNA replicon (recRNA) in tumor-free and 4T1 mammary tumor-bearing mice as a consequence of different vector administration strategies. The high potential of SFV recRNA as a biosafe approach for the development of therapeutic treatment was demonstrated. Intravenous (i.v.) inoculation of recRNA provided primary brain targeting in both tumor-free and 4T1 tumor mouse models, but local intratumoral inoculation revealed a high expression level in tumors. Moreover, we observed the predominant tumor targeting of recSFV at a reduced viral dose on i.v. and intraperitoneal (i.p.) virus inoculation, whereas the dose increase led to a broad virus distribution in mice. To prolong transgene expression, we have tested several i.v. and i.p. reinoculation strategies. A detailed evaluation of vector distribution and readministration properties could have an impact on cancer gene therapy clinical trial safety and efficacy.

AAV-mediated gene transfer of human pigment epithelium-derived factor inhibits Lewis lung carcinoma growth in mice

Pigment epithelium-derived factor (PEDF) is the most potent inhibitor of angiogenesis in the mammalian eye, and mechanisms through which PEDF exerts its antitumour activity have recently been defined. The aim of our research was to evaluate the ability of adeno-associated virus (AAV) vector-mediated transfer of human PEDF to inhibit Lewis lung carcinoma (LCC) cell growth. Intratumoural injection of AAV-PEDF caused significant reduction of the tumour volume and prolonged the survival time of mice bearing LLC cells, which were associated with decreased microvessel density and increased apoptosis in the tumours. AAV vectors represent a very promising tool for cancer gene therapy. No noticeable toxicity concerning AAV was detected as inferred from monitoring changes in animal body weight as well as basic organ structure and histological morphology, and by analyzing mouse liver and kidney function. Our findings indicate that AAV-mediated PEDF gene expression may offer an active approach to inhibit LLC growth and that treatment with AAV-PEDF may provide a promising therapeutic strategy in lung cancer treatment.