Current Gene Therapy - Volume 16, Issue 4, 2016

The ubiquitous chromatin opening element from the human HNRPA2B1-CBX3 housekeeping gene locus (A2UCOE) is able to provide stable and cell-to-cell reproducible levels of transgene expression regardless of target cell genome integration site with efficacy demonstrated in adult, embryonic and induced pluripotent stem cells and their differentiated progeny in vitro and in vivo. Here we evaluate the ability of A2UCOE-based lentiviral vectors to confer stable expression following pre-natal delivery in mice. Our results show stable post-natal A2UCOE-eGFP and A2UCOE-luciferase lentiviral vector presence in both the liver and haematopoietic system with concomitant persistence of expression demonstrating efficient transduction of both fetal liver and haematopoietic stem cells. In addition, we find that an A2UCOE-FIX lentiviral vector produces comparable amounts of plasma FIX protein to that obtained from a SFFV-FIX construct. Furthermore, the A2UCOE-FIX vector shows that at a low (0.19) average vector copy number per liver cell, it can provide stable levels of plasma FIX production, which would convert severe haemophilia B (<1%) to a mild phenotype (≈20%). Our results provide proof-ofprinciple for low dose pre-natal A2UCOE-based LV delivery to the liver as a therapeutic option for haemophilia B and potentially other metabolic conditions.

Tetracycline-regulated systems with efficient temporal and dose regulation of transgene expression are useful for development of new physiologic/pathophysiologic experimental models and gene therapy approaches. Lentiviral vectors with improved tetracycline-regulated promoters help to overcome the existing limitations such as basal activity in the drug absence, poor inducibility or unstable transgene expression. To compare conventional and improved tetracycline-regulated promoters in lentiviral based vectors in vivo, we investigated doxycycline-regulated gene transfer/expression levels in a long-term murine transplantation model and demonstrated that the lentiviral vector with the improved T11 promoter exhibited more efficient inducibility and higher gene transfer level. The time required to reverse transgene expression after doxycycline removal was increased for animals with higher gene expression levels and vector copy numbers. Examination of peripheral blood leukocytes and splenocytes revealed similar cell lineage distributions for transgene positive and negative cell populations from experimental and control mice, but increased variability in the percentages of myeloid and lymphoid cells was detected in transgene positive bone marrow cells. However, no indication of lineage bias in total bone marrow cells and no signs of hematopoietic disease were observed seven months after transplantation. Our results showed that the T11 tetracycline-regulated promoter enabled improved transgene expression in a murine transplantation model. The established system allows further development of tetracycline-regulated experimental models to investigate normal and malignant hematopoiesis.

Background: Tuberculosis (TB) is threatening disease in China and new therapeutic agents and regimens to treat TB are urgently needed. Objective: In this study, a DNA vaccine expressing Mycobacterium tuberculosis (MTB) Rv1419 antigen was constructed and its immunogenicity and therapeutic effects were evaluated. Method: Normal mice and TB model mice were immunized intramuscularly three times at two-week intervals with saline, plasmid vector pVAX1, M. vaccae vaccine, pVAX1- ag85a (rv3804c) DNA or pVAX1-rv1419 DNA, respectively. Results: At three weeks after the last immunization, flow cytometry showed a higher proportion of CD4+ T cells expressing IFN-γ (Th1) in response to Rv1419 protein in blood from the pVAX1- rv1419 DNA group compared with the saline and vector groups (P<0.05), suggesting a predominant Th1 immune response. Live bacterial loads in lungs and spleens were lower by 0.41 log10 in the pVAX1- rv1419 DNA group than in the saline controls. In addition, pathological changes in the lungs of the DNA vaccinated groups were less. These results suggest that pVAX1- rv1419 DNA could be effective for the treatment of TB, significantly increasing the Th1-type cellular immune response, and inhibiting the growth of MTB. Conclusion: Therefore pVAX1- rv1419 DNA is a candidate for inclusion in a therapeutic combination DNA vaccine against TB.

It has been reported that DOK3 protein negatively regulates LPS responses and endotoxin tolerance in mice. However, the role of DOK3 in the development of acute respiratory distress syndrome (ARDS) remains unknown. In this study, we showed that DOK3 is degraded in the lung tissues of LPS-induced ARDS. Through lentivirus transduction containing DOK3(K27R) via the intranasal route, we created a mice model, in which DOK3 maintains stable expression. We found that the forced DOK3 expression significantly attenuated LPS-induced pulmonary histological alterations, inflammatory cells infiltration, lung edema, as well as the generation of inflammatory cytokines TNFα, IL- 1β and IL-6 in BALF of LPS-induced ARDS mice. In addition, DOK3 expression apparently suppressed LPS-induced NF-κB and ERK activation. These data suggested that DOK3 expression negatively regulates the development of LPS-induced ARDS in mice.

Background: Spinal cord injury (SCI) is a serious disease which can lead to bad consequence in patients. Gene therapies, as an effective strategy, have been developed for the treatment of several diseases. But the effect for the treatment of SCI is also waiting to be practiced. Objective: Here, we explored the effect of NGF administration carried by herpes simplex virus (HSV) in the injured spinal cord. Methods: Transgenic recombinant containing human NGF was constructed by using pSP72 plasmid, then enveloped by non-replication HSV vector with deleted ICP27, ICP4 and ICP34.5 genes. Next, HSV recombinant carrying NGF was injected into cerebrospinal fluid in the lumbar cord to detect the effect of NGF for the improvement of motor function, indicated by BBB score. Meanwhile, IHC, QPCR and WB were used to confirm the NGF transduction. Results: After SCT, BBB score was largely decreased, followed by a gradual limit recovery with time going on. Q-PCR confirmed that the mRNA expression of NGF was increased in the spinal cord at 28 days post-operation, compared with that in the sham group, which suggests endogenous NGF may be available to the limit repair of motor function. Moreover, HSV carried NGF was injected into subarachnoid space of the spinal cord, which results in a significant functional improvement in hindlimbs from 7dpo to 49dpo. The level of NGF in HSV-NGF administrated group was obviously higher than that in the empty vector group and SCT group, only. Conclusion: Our results demonstrate that releasing of HSV-NGF-recombinant in subarachnoid space, can effectively improve the motor function in hindlimbs of rats subjected to SCT, which supports that strategy of HSV carrying NGF may be used for the treatment of SCI in future clinic practice.

The characteristic sequence of β-interferon matrix attachment regions (MARs) can mediate transgene expression via episomal vectors in Chinese hamster ovary (CHO) cells. However, the host cells were from hamster ovaries, which are not suitable target cells for gene therapy. In this study, we aimed to evaluate the suitability of 12 different human cell lines as target cells for gene therapy. We transfected the cells with episomal vectors and obtained colonies stably expressing the vector products after G418 screening. Therefore the stably transfected cells were split into two and further cultured either in the presence or the absence of G418. Flow cytometry was used to observe the positive rate of cell transfection and level of green fluorescent protein (GFP) expression. Plasmid rescue assays, fluorescence in situ hybridization (FISH), and fluorescence quantitative polymerase chain reaction (PCR) were used to investigate the presence and gene copy numbers of plasmid in mammalian cells. The results showed that transfection efficiency and transgene expression levels in A375, Eca-109, and Changliver cells were high. In contrast, transgene silencing was observed in BJ, HSF, and A431 cells, and low expression of the transgene was observed in the other six cell lines. In addition, the plasmid was present in the episomal state in A375, Eca-109, and Chang-liver cells with relatively low copy numbers even under nonselective conditions. Thus, our results provide the first evidence showing transgene expression of an episomal vector mediated by the characteristic motifs of MARs for maintenance of the longterm stability of episomes in different types of cells.

Although adeno-associated viral vectors have been studied for a long time, its importance as a viable gene therapy strategy has been thrusted into the limelight only in the recent years. Due to the admirable characteristics of these vectors, their potential has been thoughtfully utilized in the treatment of several neurodegenerative diseases. This mini-review focuses at recapitulating the therapeutic advances of adeno-associated viral vectors in the treatment of Parkinson’s disease by studying the various animal model experiments and clinical trials conducted since the advent of adeno-associated viral vector - based gene therapy. Additionally, the chronological analysis of the studies in the review makes it easier to understand the challenges and foretell the future prospects in this field of therapeutics.

Gene therapy emerged as a mighty alternative for conventional treatment of multiple diseases. It has been defined as a product “that mediate their effects by transcription and/or translation of transferred genetic material and/or by integrating into the host genome and that are administered as nucleic acids, viruses, or genetically engineered microorganisms. The products may be used to modify cells in vivo or transferred to cells ex vivo prior to administration to the recipient”. The first therapeutic gene therapy human trial was conducted in 1990 by Michael R. Blaese, and besides its potential, the technique suffered a major drawback after the tragical death of Jesse Gelsinger, caused by his immune response against the viral vector used in his treatment. To date, gene therapy has regained some popularity and more than 2000 clinical trials are ongoing, most of them related to the treatment or prevention of various types of cancer. Nevertheless, some types of cancer contain a rare population of stem-like cells, capable of differentiation into tumor cells, promoting the re-incidence of tumors. Those cells are generally more resilient to chemotherapy and radiotherapy and are related to tumor initiation, progression, recurrence and metastasis. The human prostate cancer (PCa) is highly heterogeneous and multifactorial, and even the markers are not precise enough to predict the clinical outcome. Furthermore, even though currently therapies can efficiently remove the tumors, the re-incidence rates are high. Gene therapy offers a handful of treatments that can halt oncogenes activation, promote the expression of suppressor genes or target cancer cells directly and induce apoptosis. Besides the risks involved, gene therapy can be of great help in the treatment of cancers and other diseases. This review aims to address the safety and potential of different gene therapy strategies used in the treatment of cancers.