Intratumoral versus Intravenous Gene Therapy Using a Transcriptionally Targeted Viral Vector in an Orthotopic Hepatocellular Carcinoma Rat Model
ABSTRACT To evaluate the feasibility of intratumoral delivery of adenoviral vector carrying a bidirectional two-step transcriptional amplification (TSTA) system to amplify transcriptional strength of cancer-specific Survivin promoter in a hepatocellular carcinoma model.
MCA-RH7777 cells were implanted in rat liver, and tumor formation was confirmed with [(18)F]fluorodeoxyglucose (18F-FDG) positron emission tomography (PET). The adenoviral vector studied had Survivin promoter driving a therapeutic gene (tumor necrosis factor-α-related apoptosis-inducing ligand [TRAIL]) and a reporter gene (firefly luciferase [FL]; Ad-pSurvivin-TSTA-TRAIL-FL). Tumor-bearing rats were administered Ad-pSurvivin-TSTA-TRAIL-FL intravenously (n = 7) or intratumorally (n = 8). For control groups, adenovirus FL under cytomegalovirus (CMV) promoter (Ad-pCMV-FL) was administered intravenously (n = 3) or intratumorally (n = 3). One day after delivery, bioluminescence imaging was performed to evaluate transduction. At 4 and 7 days after delivery, 18F-FDG-PET was performed to evaluate therapeutic efficacy.
With intravenous delivery, Ad-pSurvivin-TSTA-TRAIL-FL showed no measurable liver tumor FL signal on day 1 after delivery, but showed better therapeutic efficacy than Ad-pCMV-FL on day 7 (PET tumor/liver ratio, 3.5 ± 0.58 vs 6.0 ± 0.71; P = .02). With intratumoral delivery, Ad-pSurvivin-TSTA-TRAIL-FL showed positive FL signal from all tumors and better therapeutic efficacy than Ad-pCMV-FL on day 7 (2.4 ± 0.50 vs 5.4 ± 0.78; P = .01). In addition, intratumoral delivery of Ad-pSurvivin-TSTA-TRAIL-FL demonstrated significant decrease in tumoral viability compared with intravenous delivery (2.4 ± 0.50 vs 3.5 ± 0.58; P = .03).
Intratumoral delivery of a transcriptionally targeted therapeutic vector for amplifying tumor-specific effect demonstrated better transduction efficiency and therapeutic efficacy for liver cancer than systemic delivery, and may lead to improved therapeutic outcome for future clinical practice.
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ABSTRACT: Liver cancer is a leading cause of cancer death. Most patients are treated by arterial injection of chemoembolizing agents, providing a convenient avenue for local treatment by novel therapies, including gene therapy. Poly(beta-amino ester)s (PBAEs) were synthesized and used to form nanoparticles for non-viral transfection of buffalo rat hepatoma (MCA-RH7777) and hepatocyte (BRL-3A) lines with eGFP and luciferase DNA. Hepatoma cells were transfected with up to (98 ± 0.4)% efficacy with no measurable cytotoxicity. Hepatocytes were transfected with as high as (73 ± 0.4)% efficacy with (10 ± 4)% non-specific cytotoxicity. In contrast, positive controls (branched polyethylenimine, Lipofectamine™ 2000, and X-tremeGENE(®) DNA HP) caused 30-90% toxicity in BRL-3A cells at doses required for >50% transfection. Of the 21 optimized PBAE-DNA formulations tested, 12 showed significant specificity for hepatoma cells over hepatocytes in monoculture (p < 0.05 for both percentage transfected and eGFP expression intensity). Top polymers from eGFP studies also delivered luciferase DNA with 220 ± 30-fold and 470 ± 30-fold greater specificity for hepatoma cells than hepatocytes. Transfections of co-cultures of hepatoma and hepatocytes with eGFP DNA also showed high specificity (1.9 ± 0.1- to 5.8± 1.4-fold more transfected hepatoma cells than hepatocytes, measured by percentage transfected and flow cytometry). By eGFP intensity, up to 530 ±60-fold higher average expression per cell was measured in hepatoma cells. One top formulation caused (95 ± 0.2)% transfection in hepatoma cells and (27 ± 0.2)% in hepatocytes [(96 ± 9)% relative hepatocyte viability]. PBAE-based nanoparticles are a viable strategy for liver cancer treatment, delivering genes to nearly 100% of cancer cells while maintaining high biomaterial-mediated specificity to prevent toxic side-effects on healthy hepatocytes. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.Journal of Biomedical Materials Research Part A 04/2013; 101(7). DOI:10.1002/jbm.a.34616 · 2.83 Impact Factor
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ABSTRACT: In hepatocellular carcinoma (HCC), tumor specificity of gene therapy is of utmost importance to preserve liver function. MicroRNAs (miRNAs) are powerful negative regulators of gene expression and many are downregulated in human HCC. We identified seven miRNAs that are also downregulated in tumors in a rat hepatoma model (P<0.05) and attempted to improve tumor specificity by constructing a panel of luciferase-expressing vectors containing binding sites for these miRNAs. Attenuation of luciferase expression by the corresponding miRNAs was confirmed across various cell lines and in mouse liver. We then tested our vectors in tumor-bearing rats and identified two miRNAs, miR-26a and miR-122, that significantly decreased expression in liver compared with the control vector (6.40 and 0.26%, respectively; P<0.05). In tumor, miR-122 had a nonsignificant trend towards decreased (∼50%) expression, whereas miR-26 had no significant effect on tumor expression. To our knowledge, this is the first work using differentially expressed miRNAs to de-target transgene expression in an orthotopic hepatoma model and to identify miR-26a, in addition to miR-122, for de-targeting liver. Considering the heterogeneity of miRNA expression in human HCC, this information will be important in guiding development of more personalized vectors for the treatment of this devastating disease.Gene Therapy advance online publication, 30 May 2013; doi:10.1038/gt.2013.24.Gene therapy 05/2013; DOI:10.1038/gt.2013.24 · 4.20 Impact Factor
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ABSTRACT: A gene delivery system was designed to carry a payload to integrin overexpressing cells. Branched-polyethyleneimine (bPEI) condensed plasmid DNA was encapsulated into targeted stealth liposomes, thereby combining the condensing and transfection properties of bPEI with the stealth and targeting properties of the liposomal carrier system. PR_b was used as a targeting ligand – a peptide we designed to bind specifically to the cancer cell surface marker α5β1 integrin – and such a robust receptor–ligand interaction achieved higher specificity than what has been previously reported for targeted delivery systems. In the process of formulating the PR_b functionalized gene delivery vehicle, we developed a protocol to fully encapsulate condensed DNA in liposomes and accurately quantify the total DNA in the system. We demonstrate that compared to non-targeted stealth liposomes and non-encapsulated condensed DNA, the PR_b functionalized stealth liposomes mediated improved in vitro transfection specifically to colon cancer cells overexpressing the α5β1 integrin. Furthermore, when administered in vivo to metastatic tumor bearing mice, PR_b functionalized stealth liposomes outperformed non-targeted liposomes and delivered genes specifically to the tumor site.12/2012; 1(4). DOI:10.1039/C2BM00128D