Current Gene Therapy - Volume 15, Issue 3, 2015

Activation of hepatic stellate cells (HSCs) is a key event in pathogenesis of liver fibrosis and represents an orchestral interplay of inhibiting and activating transcription factors like forkhead box f1 (Foxf1), being described to stimulate pro-fibrogenic genes in HSCs. Here, we evaluated a lipidbased liver-specific delivery system (DBTC) suitable to transfer Foxf1 siRNA specifically to HSCs and examined its antifibrotic potential on primary HSCs and LX-2 cells as well as in a murine model of bile duct ligation (BDL)-induced secondary cholestasis. Foxf1 silencing reduced proliferation capacity and attenuated contractility of HSCs. Systemic administration of DBTC-lipoplexes in mice was sufficient to specifically silence genes expressed in different liver cell types. Using intravital and immunofluorescence microscopy we confirmed the specific delivery of Cy3-labeled DBTC to the liver, and particularly to HSCs. Repeated treatment with DBTC-lipoplexes resulted in siRNA-mediated silencing of Foxf1 early after BDL and finally attenuated progression of the fibrotic process. Decreased HSC activation in-effect ameliorated liver injury as shown by substantial reduction of necrotic area and deposition of extracellular matrix. Our findings suggest that Foxf1 may serve as a target gene to disrupt progression of liver fibrosis and DBTC might provide a potentially feasible and effective tool for HSC-specific delivery of therapeutic RNA.

New targets and therapeutic approaches for vascular targeted strategies in oncology are continuously explored. Endoglin, a co-receptor of TGF-β, is a known target, however, its silencing with vector-based RNA interference technology has not been evaluated yet. Therefore, in our study, we assembled plasmid DNA coding for shRNA against endoglin, and used gene electrotransfer as a delivery method to determine its antitumor and vascular targeted effects. In vitro and in vivo data provide evidence of vascular targeted effects of endoglin silencing. The vascular targeted action of endoglin silencing could be described as a result of two separated effect; antiangiogenic and vascular disrupting effect. This was first supported by in vitro data; predominantly by reduction of proliferation and tube formation of endothelial cells. In the TS/A murine mammary carcinoma model, in which the tumor cells do not express endoglin, reduced tumor growth and number of vessels were observed. Quick destruction of existing activated blood vessels at the site of tumor cells’ injection and sustained growth of tumors afterwards was observed in tumors that were growing in dorsal window chamber by intravital microscopy. This observation supports both vascular disrupting and antiangiogenic action. In conclusion, the results of our study provide evidence of endoglin as a valid target for cancer therapy and support further development of plasmid shRNA delivery, which have prolonged antitumor effect, especially in combined schedules.

Wiskott-Aldrich syndrome (WAS) is a life-threatening disorder characterized by immunodeficiency, thrombocytopenia and eczema. Hematopoietic stem cell gene therapy can cure WAS but remains associated with the risk of leukemogenesis. In an effort to decrease the risk of gene-therapy induced leukemia associated with the use of first generation gamma-retroviral vectors, we have developed a series of codon-optimized WASP-expressing alpha-, gammaretro- and lentiviral selfinactivating vectors and have performed comparative studies with regard to expression, vector dose and genotoxicity in vitro. In comparison to the conventional LTR-driven gammaretroviral vector, the self-inactivating (SIN) Elongation Factor 1 short form (EFS) - driven vectors presented decreased expression potency per vector copy. However, the safety profile of SIN vectors was superior, as evidenced by decreased genotoxicity in a murine hematopoietic progenitor cell - based in vitro immortalization assay. Using an in vitro OP9 stroma cell - based co-culture system, we provide evidence that a codon-optimized lentiviral vector improves the B-cell differentiation block characteristic of WAS. Taken together, our studies represent a further step towards the development of a novel codon-optimized coWASP SIN vector for ex-vivo HSC gene therapy for Wiskott-Aldrich syndrome.

Rare genetic diseases account for a considerable amount of fatalities and even their ‘mild’ or ‘non-lethal’ forms can produce drastic and undesirable discomfort to affected individuals. Various gene therapeutic approaches were tested for developing novel therapeutic concepts to treat these genetic diseases. Sleeping Beauty (SB) transposase represents one of these gene therapeutic systems which can be utilized for stable phenotypic correction. It is a transposable element which was resurrected and optimized for transposing genetic elements resulting in somatic integration of the transgene. Because of its versatile activity in many different organs, SB transposase has been explored for ex-vivo gene delivery and in vivo gene delivery including recently launched clinical trials based on engineered T-cells for tumor therapy and approaches to treat retinal degenerations. Here we will provide a state-of-the-art overview of preclinical studies for treatment of rare genetic diseases based on the SB transposase system for stable correction of the genetic defect. In this review, diseases affecting the blood system, the connective tissue, the immune system, the metabolism, and the nervous system and their treatment utilizing the SB transposase system will be discussed. Moreover, advantages and disadvantages of SB transposase-based gene therapeutic approaches will be mentioned. Although improvements of the SB transposase systems regarding genotoxicity and efficient delivery especially for applications in large mammals are desirable, the SB transposase system remains to hold great promise for curing rare genetic disease.

Amyotrophic lateral sclerosis (ALS) is an incurable, chronic, fatal neuro-degenerative disease characterized by progressive loss of moto-neurons and paralysis of skeletal muscles. Reactivating dysfunctional areas is under earnest investigation utilizing various approaches. Here we present an innovative gene-cell construct aimed at reviving inert structure and function. Human umbilical cord blood cells (hUCBCs) transduced with adeno-viral vectors encoding human VEGF, GDNF and/or NCAM genes were transplanted into transgenic ALS mice models. Significant improvement in behavioral performance (open-field and grip-strength tests), as well as increased life-span was observed in rodents treated with NCAM-VEGF or NCAM-GDNF co-transfected cells. Active trans-gene expression was found in the spinal cord of ALS mice 10 weeks after delivering genetically modified hUCBCs, and cells were detectable even 5 months following transplantation. Our gene-cell therapy model yielded prominent symptomatic control and prolonged life-time in ALS. Incredible survivability of xeno-transpanted cells was also observed without any immune-suppression. These results suggest that engineered hUCBCs may offer effective gene-cell therapy in ALS.

The feasibility of utilising bacteria as vectors for gene therapy is becoming increasingly recognised. This is primarily due to a number of intrinsic properties of bacteria such as their tumour targeting capabilities, their ability to carry large genetic or protein loads and the availability of wellestablished genetic engineering tools for a range of common lab strains. However, a number of issues relating to the use of bacteria as vectors for gene therapy need to be addressed in order for the field to progress. Amongst these is the need for the development of non-invasive detection/imaging systems for bacteria within a living host. In vivo optical imaging has advanced preclinical research greatly, and typically involves engineering of bacteria with genetic expression constructs for luminescence (e.g. the lux operon) or fluorescent proteins (GFP etc.). This requirement for genetic modification can be restrictive, where engineering is not experimentally appropriate or technologically feasible (e.g. due to lack of suitable engineering tools). We describe a novel strategy exploiting endogenous bacterial enzymatic activity to specifically activate an exogenously administered fluorescent imaging probe. The red shifted, quenched fluorophore CytoCy5S is reduced to a fluorescent form by bacterial-specific nitroreductase (NTR) enzymes. NTR enzymes are present in a wide range of bacterial genera and absent in mammalian systems, permitting highly specific detection of Gram-negative and Gram–positive bacteria in vivo. In this study, dose-responsive bacterial-specific signals were observed in vitro from all genera examined – E. coli, Salmonella, Listeria, Bifidobacterium and Clostridium difficile. Examination of an NTR-knockout strain validated the enzyme specificity of the probe. In vivo whole-body imaging permitted specific, dose-responsive monitoring of bacteria over time in various infection models, and no toxicity to bacteria or host was observed. This study demonstrates the concept of exploiting innate NTR activity as a reporting strategy for wild-type bacteria using optical imaging, while the concept may also be extended to NTR-specific probes for use with other imaging modalities.

The genetic engineering of T cells can lead to enhanced immune-mediated tumour destruction and harbors a great potential for the treatment of cancer. Recent efforts have centered on the design of receptors to re-direct the specificity of T cells towards tumour antigens by means of viral gene transfer. This strategy has shown great success in a number of phase one clinical trials. However, there are still challenges to overcome. On the one hand, T cell function can be further improved to optimize the therapeutic outcome. On the other hand, so called safety switches are required to deal with possible on and off target toxicities. In this review, we will give a brief summary of the success and risks of T cell gene therapy before discussing in detail current strategies to enhance effector function, persistence and safety of adoptively transferred T cells.

Epithelial Mesenchymal Transition (EMT) is an event where epithelial cells acquire mesenchymal- like phenotype. EMT can occur as a physiological phenomenon during tissue development and wound healing, but most importantly, EMT can confer highly invasive properties to epithelial carcinoma cells. The impairment of E-cadherin expression, an essential cell-cell adhesion protein, together with an increase in the expression of mesenchymal markers, such as N-cadherin, vimentin, and fibronectin, characterize the EMT process and are usually correlated with tumor migration, and metastization. A wide range of micro-environmental and intracellular factors regulate tumor development and progression. The dynamic cross-talk between the adhesion-related proteins such as E-cadherin and the EMT-related transcription factors, with special focus on TWIST, will be discussed here, with the aim of finding a suitable biological pathway to be used as potential target for cancer therapy. Emerging concepts such as the role of the PI3K/AKT/TWIST pathway in the regulation of the E-cadherin expression will be highlighted, since it seems to be consistently involved in cells EMT. The wellknown efficacy of the RNA interference as a tool to silence the expression of specific proteins has come into focus as a strategy to control different tumor sub-populations. Despite the oligonucleotides enormous sensitivity and low in vivo stability, new (nano)technological solutions are expected to enable RNAi clinical application in cancer therapy.

Therapeutic revascularization had been considered as the most potential strategy for treating ischemic diseases. Reconstruction of mature blood vessels, which is the key for functional revascularization, is a complex process involving multiple angiogenesis factors. Attempts had been made to promote functional revascularization by delivering vectors or other macromolecules that could positively regulate angiogenesis; however, the delivery method of these therapeutic angiogenesis factors had been mostly limited to direct intramuscular injection. In this study, we showed that compared to intramuscular injection, the hydrodynamic limb vein (HLV) injection of naked shorthairpin RNA expression plasmid targeting PHD2 (shPHD2) into critical himblimb ischemia mice could increase not only the expressions of HIF-dependent and HIF-independent angiogenic factors, but also tissue protective factors involved in various endogenous pathways more efficiently. We also found that PHD2-silencing enhanced innate endogenous recovery mechanism, as the expression levels of these factors had been slightly upregulated merely by the ischemic condition. Finally, we showed that HLV injection of shPHD2 promoted the formation of mature and functional vessels, and thus, enhances the recovery of ischemic hindlimbs more efficiently. These results suggest that HLV delivery of shPHD2 might become a promising treatment strategy to promote vascular regeneration in critical limb ischemia disease via enhancing innate endogenous pathways.