Current Gene Therapy (v.12, #6)

Growth Responses Following a Single Intra-Muscular hGH Plasmid Administration Compared to Daily Injections of hGH in Dwarf Mice by Eliza Higuti, Claudia R. Cecchi, Nelio A.J. Oliveira, Daniel P. Vieira, Thomas G. Jensen, Alexander A.L. Jorge, Paolo Bartolini, Cibele N. Peroni (437-443).
In previous work, sustained levels of circulating human growth hormone (hGH) and a highly significant weight increase were observed after electrotransfer of naked plasmid DNA (hGH-DNA) into the muscle of immunodeficient dwarf mice (lit/scid). In the present study, the efficacy of this in vivo gene therapy strategy is compared to daily injections (5&#x3BC;g/twice a day) of recombinant hGH (r-hGH) protein, as assessed on the basis of several growth parameters. The slopes of the two growth curves were found to be similar (P > 0.05): 0.095 g/mouse/d for protein and 0.094 g/mouse/d for DNA injection. In contrast, the weight increases averaged 35.5% (P < 0.001) and 23.1% (P < 0.01) for protein and DNA administration, respectively, a difference possibly related to the electroporation methodology. The nose-to-tail linear growth increases were 15% and 9.6% for the protein and DNA treatments, respectively, but mouse insulin-like growth factor I (mIGF-I) showed a greater increase over the control with DNA (5- to 7-fold) than with protein (3- to 4-fold) administration. The weight increases of several organs and tissues (kidneys, spleen, liver, heart, quadriceps and gastrocnemius muscles) were 1.3- to 4.6-fold greater for protein than for DNA administration, which gave a generally more proportional growth. Glucose levels were apparently unaffected, suggesting the absence of effects on glucose tolerance. A gene transfer strategy based on a single hGH-DNA administration thus appears to be comparable to repeated hormone injections for promoting growth and may represent a feasible alternative for the treatment of growth hormone deficiency.

Induction of Apoptosis and Sensitization of Head and Neck Squamous Carcinoma Cells to Cisplatin by Targeting Survivin Gene Expression by Zakir Khan, Ram P. Tiwari, Noor Khan, Godavarthi B.K.S. Prasad, Prakash S. Bisen (444-453).
Survivin is known to be highly-expressed in various carcinomas; and is associated with their biologically aggressive characteristics and drug resistance. We have previously reported survivin as an important anti-apototic protein involved in head and neck squamous cell carcinoma (HNSCC) tumorigenesis and providing resistance to conventional cancer therapies. The purpose of present study was to investigate the potential of survivin as a therapeutic target in HNSCC. This study was designed to explore the effect(s) of survivin-siRNA therapy on the apoptosis in HNSCC cells, and its influence on cisplatin-sensitivity. Lentivirus vector was developed to deliver survivin specific siRNA into cancer cells. The data indicates that silencing of survivin-mediated by Lentivirus-siRNA therapy effectively suppressed cancer cell proliferation and induced caspase-dependent apoptosis in HNSCC cells. The study also shows that the response of HNSCC cells to cisplatin drug treatment at clinically relevant level was limited. We observed survivin to be a key factor involved in this cisplatin-resistance mechanism, and down-regulation of survivin significantly increased sensitivity of cancer cells to cisplatin-mediated apoptosis. Thus, this combination therapy acts as a multimodality regimen with significant potential to improve clinical outcomes in head and neck cancers.

In Vivo, Cardiac-Specific Knockdown of a Target Protein, Malic Enzyme- 1, in Rat via Adenoviral Delivery of DNA for Non-Native miRNA by J. Michael O&#x2019;Donnell, Asha Kalichira, Jian Bi, Edward D. Lewandowski (454-462).
This study examines the feasibility of using the adenoviral delivery of DNA for a non-native microRNA to suppress expression of a target protein (cytosolic NADP+-dependent malic-enzyme 1, ME1) in whole heart in vivo, via an isolated-heart coronary perfusion approach. Complementary DNA constructs for ME1 microRNA were inserted into adenoviral vectors. Viral gene transfer to neonatal rat cardiomyocytes yielded 65% suppression of ME1 protein. This viral package was delivered to rat hearts in vivo (Adv.miR_ME1, 1013 vp/ml PBS) via coronary perfusion, using a cardiacspecific isolation technique. ME1 mRNA was reduced by 73% at 2-6 days post-surgery in heart receiving the Adv.miR_ME1. Importantly, ME1 protein was reduced by 66% (p < 0.0002) at 5-6 days relative to sham-operated control hearts. Non-target protein expression for GAPDH, calsequestrin, and mitochondrial malic enzyme, ME3, were all unchanged. The non-target isoform, ME2, was unchanged at 2-5 days and reduced at day 6. This new approach demonstrates for the first time significant and acute silencing of target RNA translation and protein content in whole heart, in vivo, via non-native microRNA expression.

Gene Therapy in Liver Diseases: State-of-the-Art and Future Perspectives by Kalliopi Domvri, Paul Zarogoulidis, Konstantinos Porpodis, Maria Koffa, Maria Lambropoulou, Stylianos Kakolyris, George Kolios, Konstantinos Zarogoulidis, Ekaterini Chatzaki (463-483).
Gene therapy is a fundamentally novel therapeutic approach that involves introducing genetic material into target cells in order to fight or prevent disease. A number of different strategies of gene therapy are tested at experimental and clinical levels, including: a) replacing a mutated gene that causes disease with a healthy copy of the gene, b) inactivating a mutated gene that its improper function causes pathogenesis, c) introducing a new gene coding a therapeutic compound to fight a disease, d) introducing to the target organ an enzyme converting an inactive pro-drug to its cytotoxic metabolite. In gene therapy, the transcriptional machinery of the patient is used to produce the active factor that exerts the intended therapeutic effect, ideally in a permanent, tissue-specific and manageable way. The liver is a major target for gene therapy, presenting inherited metabolic defects of single-gene etiology, but also severe multifactorial pathologies with limited therapeutic options such as hepatocellular carcinoma. The initial promising results from gene therapy strategies in liver diseases were followed by skepticism on the actual clinical value due to specificity, efficacy, toxicity and immune limitations, but are recently re-evaluated due to progress in vector technology and monitoring techniques. The significant amount of experimental data along with the available information from clinical trials are systematically reviewed here and presented per pathological entity. Finally, future perspectives of gene therapy protocols in hepatology are summarized.

Therapeutic Effect of Ribbon-Type Nuclear Factor-&#x3BA;B Decoy Oligonucleotides in a Rat Model of Inflammatory Bowel Disease by Kazunari Ozaki, Hirofumi Makino, Motokuni Aoki, Takashi Miyake, Natsuki Yasumasa, Mariana Kiomy Osako, Hironori Nakagami, Hiromi Rakugi, Ryuichi Morishita (484-492).
Background: The pathogenesis of inflammatory bowel disease (IBD) involves local expression of inflammatory cytokines, some of which are coordinated by nuclear factor-&#x3BA;B (NF-&#x3BA;B). Several reports documented the therapeutic potential of double-stranded phosphorothioated decoy oligonucleotides (S-ODNs) targeting NF-&#x3BA;B in IBD models. However, S-ODNs are easily degraded by endonucleases. In this study, we employed newly developed nonchemically modified ribbon-type NF-&#x3BA;B decoy ODNs (R-ODNs) with loop ends that increase the stability, and investigated their therapeutic effect in rats with dextran sulfate sodium (DSS)-induced colitis. Methodology/Principal Findings: We administered R-ODN, S-ODN, or scrambled ODN (Scr-ODN) to rats with DSSinduced IBD using ultrasound with contrast microbubbles to enhance the transfection efficiency of ODN. Until day 10 after DSS treatment, the rats showed a decrease in body weight and survival rate and an increase in the disease activity index (DAI). In rats treated with S-ODN or R-ODN, the survival rate, colon length, and DAI were significantly improved. In addition, DSS-induced expression of tumor necrosis factor-&#x3B1;, interleukin (IL)-6, and IL-1β was significantly decreased. Of importance, treatment with R-ODN was more effective to improve disease conditions as compared to S-ODN. Conclusions: These data suggest that intracolonic administration of R-ODN may be effective to treat DSS-induced colitis.

Genetical modification of T lymphocytes by chimeric antigen receptor (CAR), which has the affinity to tumor associated antigen (TAA), can redirect the normal T lymphocytes with tumor specificity. Through optimization of the CAR construct from first generation to third generation, the properties of these CAR-modified T lymphocytes, including, cytokines release, proliferative capacity, in vivo survival time, have been remarkably improved. These improvements accelerate the clinical applications of the CAR-modified T lymphocytes. Due to CD19, and CD20 specificity, CARmodified T lymphocytes have been clinically used to treat leukemia. Notably, T lymphocytes genetically engineered by Carl June group with CAR targeting to CD19 have expanded more than 1,000 folds by clearing all CD19 positive leukemia cells, even the normal B cells, which infiltrated in bone marrow, and survived for more than 6 months. This encouraging report demonstrates the overwhelming tumor-lytic ability of the CAR-modified T lymphocytes; it also raises the concern about the chronic toxicity. Here, we emphasize the safety concern when using the CAR-modified T lymphocytes. We also summarize strategies exploited to clear the genetically engineered T lymphocytes under an urgent condition, which increases the safety and optimizes the applications of the CAR-modified T lymphocytes.