Current Gene Therapy (v.15, #5)

CFTR Inactivation by Lentiviral Vector-mediated RNA Interference and CRISPR-Cas9 Genome Editing in Human Airway Epithelial Cells by Jessica Bellec, Marc Bacchetta, Davide Losa, Ignacio Anegon, Marc Chanson, Tuan Huy Nguyen (447-459).
Background: Polarized airway epithelial cell cultures modelling Cystic Fibrosis Transmembrane conductance Regulator (CFTR) defect are crucial for CF and biomedical research. RNA interference has proven its value to generate knockdown models for various pathologies. More recently, genome editing using CRISPR-Cas9 artificial endonuclease was a valuable addition to the toolbox of gene inactivation. Methods: Calu-3 cells and primary HAECs were transduced with HIV-1-derived lentiviral vectors (LVV) encoding small hairpin RNA (shRNA) sequence or CRISPR-Cas9 components targeting CFTR alongside GFP. After sorting of GFP-positive cells, CFTR expression was measured by RT-qPCR and Western blot in polarized or differentiated cells. CFTR channel function was assessed in Ussing chambers. Il-8 secretion, proliferation and cell migration were also studied in transduced cells. Results: shRNA interference and CRISPRCas9 strategies efficiently decreased CFTR expression in Calu-3 cells. Strong CFTR knockdown was confirmed at the functional level in CRISPR-Cas9-modified cells. CFTR-specific shRNA sequences did not reduce gene expression in primary HAECs, whereas CRISPR-Cas9-mediated gene modification activity was correlated with a reduction of transepithelial secretion and response to a CFTR inhibitor. CFTR inactivation in the CRISPR-Cas9-modified Calu-3 cells did not affect migration and proliferation but slightly increased basal interleukin-8 secretion. Conclusion: We generated CFTRinactivated cell lines and demonstrated that CRISPR-Cas9 vectorised in a single LVV efficiently promotes CFTR inactivation in primary HAECs. These results provide a new protocol to engineer CF primary epithelia with their isogenic controls and pave the way for manipulation of CFTR expression in these cultures.

Heart tissue has a diminished ability to repair after myocardial infarction (MI). Bone marrow- derived mesenchymal stem cells (BMSCs) have been used effectively to heal damaged tissue after MI. Hypoxia-inducible factor-1α (HIF-1α) can induce transcription of numerous pro-angiogenic genes and enhance stem cell survival. Here, we investigated whether HIF-1α-transduced BMSCs could enhance tissue repair after MI, and compared the value of micro-PET/CT and echocardiography for evaluation of therapeutic effects. Rat BMSCs were transduced with a lentivirus expressing HIF-1α and NIS (Lenti-HIF-1α-NIS). Sodium iodide symporter (NIS) functioned as effective reporter gene, allowing monitoring of BMSCs transplanted into the rat heart for up to 2 weeks using micro-SPECT/CT imaging. In a rat MI model, after transplantation of HIF-1α-NIS-transduced BMSCs to the MI zone, more expression of HIF-1α,VEGF and Ang-4, more improvement of metabolism, less fibrotic tissue and cardiomyocyte apoptosis were detected in the MI zone. Moreover, we found that most of the transplanted HIF-1α-NIS-transduced BMSCs differentiated into endothelial cells, and engineered new blood vessels in MI zone. Metabolic activity significantly increased at an early time point (2 weeks after transplantation) and lead to a sustained increase (4 weeks), as indicated by 18F-FDG uptake in micro-PET/CT imaging. Echocardiography indicated no improvement in cardiac function at 2 weeks and small improvement at 4 weeks. This study indicated that 18F-FDG micro-PET/CT was more useful for evaluating early therapeutic effects than echocardiography.

Folic acid (FA) has high affinity to folate receptors (FRs), which have three isoforms: FRα, FRβ and FRγ. Among them, FRα is a tumor specific receptor, as it is frequently over-expressed in diverse malignancies but not in normal tissues. In this study, we have conjugated FA to a chitosan-poly(ethylenimine) copolymer, and have confirmed the low cytotoxicity of the product (namely “CP1.3K-FA”) in cancer cells. The transfection efficiency of CP1.3K-FA has been shown by the EGFP transfection assay to be higher than that of the unmodified chitosan-poly(ethylenimine) copolymer under optimal conditions. Results of the luciferase activity assay have also indicated that the transfection efficiency of CP1.3K-FA is comparable to that of Fugene HD in B16 and U87 cells. Our results have suggested that CP1.3K-FA warrants further development as a vector for gene delivery in cancer cells.

Biodistribution of LV-TSTA Transduced Rat Bone Marrow Cells Used for “Ex-vivo” Regional Gene Therapy for Bone Repair by Farhang Alaee, Cynthia Bartholomae, Osamu Sugiyama, Mandeep S. Virk, Hicham Drissi, Qian Wu, Manfred Schmidt, Jay R. Lieberman (481-491).
“Ex vivo” regional gene therapy using lentiviral (LV) vectors to over-express bone morphogenetic protein 2 (BMP-2) is an effective way to enhance bone healing in animal models. Here, we evaluated two different “ex vivo” approaches using either “same day” rat bone marrow cells (SDRBMCs) or cultured rat bone marrow cells (C-RBMCs), both transduced with a LV based two-step transcriptional activation system overexpressing GFP (LV-TSTA-EGFP), to assess the fate of the transduced cells and the safety of this approach. The transduced cells were implanted in femoral defects of syngeneic rats. Animals were sacrificed at 4, 14, 28 and 56 days after surgery (n=5 per group). Viral copies were detectable in the defect site of SD-RBMC group and gradually declined at 8w (5 log decrease compared to 4d). In the C-RBMC animals, there was a 2-4 log decline in the viral copy numbers at 2w and 4w, but at 8w there was a relative rise (about 100 fold) in the number of the viral vectors in the defect site of 4 (out of 5) animals compared to the previous time points. For both gene transfer approaches, the pattern of tissue distribution was non-specific and no histological abnormalities were noted in either group. In summary, we demonstrated that the LV-TSTA transduced cells remain in the defect site for at least 56 days, though the numbers decreased over time. There were no consistent findings of viral copies in internal organs which is encouraging with respect to the development of this strategy for use in humans.

Bifunctional siRNA Containing Immunostimulatory Motif Enhances Protection Against Pandemic H1N1 Virus Infection by Gaurav Joshi, Paban Kumar Dash, Ankita Agarwal, Shashi Sharma, Manmohan Parida (492-502).
Rationale: The first influenza pandemic of 21st century was attributed to a novel quadruple reassortant H1N1 virus that emerged in 2009. Currently available therapies for influenza have drugresistant. Therefore, there is a need to develop new generation immunotherapeutic antiviral strategy. This study described the efficacy of a novel bifunctional immunostimulatory siRNAs against H1N1pdm swine flu virus by targeting the Nucleocapsid (NP) gene. Methods & Findings: Small interfering RNAs (siRNA) targeting conserved region of NP were screened for antiviral efficacy in human lung epithelial cells (A549). Further, a bifunctional siRNA was synthesized by combining immunostimulatory sequence (5'-UGUGU-3') with NP specific siRNA. This immunostimulatory siRNA (NP-1-is) revealed strong antiviral effect through reduction in mRNA copies (99.58%), reduction in virus associated cell apoptosis and inhibition of nucleocapsid protein in western blot. This immunostimulatory siRNA was found more effective than nontagged siRNA. Further studies including dose dependent and time course kinetics revealed that the NP-1-is siRNA is more effective at 20-80 nM with significant protection upto 48 hpi. Besides, the qRT-PCR and western blot analysis confirmed higher antiviral response of immunostimulatory siRNA was due to upregulation of TLR-7 MyD88, IRF-7 and IFN-α. Conclusions: This study paves the way for broad-spectrum RNAi-based therapeutics using immunostimulatory motif towards improved antiviral effect. Hence this approach will be useful to confront the sudden emergence of pandemic strains.

Therapy for Dominant Inherited Diseases by Allele-Specific RNA Interference: Successes and Pitfalls by Delphine Trochet, Bernard Prudhon, Stéphane Vassilopoulos, Marc Bitoun (503-510).
RNA interference (RNAi) is a conserved mechanism for post-transcriptional gene silencing mediated by messenger RNA (mRNA) degradation. RNAi is commonly induced by synthetic siRNA or shRNA which recognizes the targeted mRNA by base pairing and leads to target-mRNA degradation. RNAi may discriminate between two sequences only differing by one nucleotide conferring a high specificity of RNAi for its target mRNA. This property was used to develop a particular therapeutic strategy called “allele-specific-RNA interference” devoted to silence the mutated allele of genes causing dominant inherited diseases without affecting the normal allele. Therapeutic benefit was now demonstrated in cells from patients and animal models, and promising results of the first phase Ib clinical trial using siRNA-based allele-specific therapy were reported in Pachyonychia Congenita, an inherited skin disorder due to dominant mutations in the Keratin 6 gene. Our purpose is to review the successes of this strategy aiming to treat dominant inherited diseases and to highlight the pitfalls to avoid.

Current Genome Editing Tools in Gene Therapy: New Approaches to Treat Cancer by Oleg Shuvalov, Alexey Petukhov, Alexandra Daks, Olga Fedorova, Alexander Ermakov, Gerry Melino, Nickolai A Barlev (511-529).
Gene therapy suggests a promising approach to treat genetic diseases by applying genes as pharmaceuticals. Cancer is a complex disease, which strongly depends on a particular genetic make-up and hence can be treated with gene therapy. From about 2,000 clinical trials carried out so far, more than 60% were cancer targeted. Development of precise and effective gene therapy approaches is intimately connected with achievements in the molecular biology techniques. The field of gene therapy was recently revolutionized by the introduction of “programmable” nucleases, including ZFNs, TALENs, and CRISPR, which target specific genomic loci with high efficacy and precision. Furthermore, when combined with DNA transposons for the delivery purposes into cells, these programmable nucleases represent a promising alternative to the conventional viral-mediated gene delivery. In addition to “programmable” nucleases, a new class of TALE- and CRISPR-based “artificial transcription effectors” has been developed to mediate precise regulation of specific genes. In sum, these new molecular tools may be used in a wide plethora of gene therapy strategies. This review highlights the current status of novel genome editing tools and discusses their suitability and perspectives in respect to cancer gene therapy studies.