Current Gene Therapy (v.14, #5)

Single-Stranded γPNAs for In Vivo Site-Specific Genome Editing via Watson-Crick Recognition by Raman Bahal, Elias Quijano, Nicole A. McNeer, Yanfeng Liu, Dinesh C. Bhunia, Francesco Lopez-Giraldez, Rachel J. Fields, William M. Saltzman, Danith H. Ly, Peter M. Glazer (331-342).
Triplex-forming peptide nucleic acids (PNAs) facilitate gene editing by stimulating recombination of donorDNAs within genomic DNA via site-specific formation of altered helical structures that further stimulate DNA repair.However, PNAs designed for triplex formation are sequence restricted to homopurine sites. Herein we describe a novelstrategy where next generation single-stranded gamma PNAs (γPNAs) containing miniPEG substitutions at the gammaposition can target genomic DNA in mouse bone marrow at mixed-sequence sites to induce targeted gene editing. In additionto enhanced binding, γPNAs confer increased solubility and improved formulation into poly(lactic-co-glycolic acid)(PLGA) nanoparticles for efficient intracellular delivery. Single-stranded γPNAs induce targeted gene editing at frequenciesof 0.8% in mouse bone marrow cells treated ex vivo and 0.1% in vivo via IV injection, without detectable toxicity.These results suggest that γPNAs may provide a new tool for induced gene editing based on Watson-Crick recognitionwithout sequence restriction.

Tau Silencing by siRNA in the P301S Mouse Model of Tauopathy by Hong Xu, Thomas W. Rosler, Thomas Carlsson, Anderson de Andrade, Ondrej Fiala, Matthias Hollerhage, Wolfgang H. Oertel, Michel Goedert, Achim Aigner, Gunter U. Hoglinger (343-351).
Suppression of tau protein expression has been shown to improve behavioral deficits in mouse models oftauopathies, offering an attractive therapeutic approach. Experimentally this had been achieved by switching off the promoterscontrolling the transgenic human tau gene (MAPT), which is not possible in human patients. The aim of the presentstudy was therefore to evaluate the effectiveness of small interfering RNAs (siRNAs) and their cerebral delivery to suppresshuman tau expression in vivo, which might be a therapeutic option for human tauopathies. We used primary corticalneurons of transgenic mice expressing P301S-mutated human tau and Lund human mesencephalic (LUHMES) cells tovalidate the suppressive effect of siRNA in vitro. For measuring the effect in vivo, we stereotactically injected siRNA intothe brains of P301S mice to reveal the suppression of tau by immunochemistry (AT180, MC1, and CP13 antibodies). Wefound that the Accell SMART pool siRNA against MAPT can effectively suppress tau expression in vitro and in vivowithout a specific delivery agent. The siRNA showed a moderate distribution in the hippocampus of mice after single injection.NeuN, GFAP, Iba-1, MHC II immunoreactivities and the terminal deoxynucleotidyl transferase dUTP nick endlabeling (TUNEL) assay showed neither signs of neurotoxicity or neuroinflammation nor apoptosis when MAPT siRNA ispresent in the hippocampus. Our data suggest that siRNA against MAPT can serve as a potential tool for gene therapy intauopathies.

Integrase-Deficient Lentivirus: Opportunities and Challenges for Human Gene Therapy by Kuan-Can Liu, Bao-Shun Lin, An-Ding Gao, Hong-Yu Ma, Meng Zhao, Rui Zhang, Hui-Hui Yan, Xun-Fei Yi, Si-Jie Lin, Jian-Wen Que, Xiao-Peng Lan (352-364).
Lentiviruses are powerful tools for gene delivery and have been widely used for the dissection of gene functionsin both replicating and quiescent cells. Recently, lentiviruses have also been used for delivering target sequences ingene therapy. Although the lentiviral system provides sustained exogenous gene expression, serious concerns have beenraised due to its unfavorable insertion-mediated mutagenesis effect, thereby resulting in the silencing or activation ofsome unexpected genes. Thus, an array of modifications of the original vectors may reduce risks. Here, we briefly reviewthe structure of the integrase protein, which is an essential protein for viral insertion and integration; the mechanisms ofintegrase-mediated integration; and the effects of the modifications of integrase. Moreover, we discuss the advantages resultingfrom integrase modifications and their future applications. Taken together, the generation of integrase-deficientlentivirus (IDLV) not only provides us with an opportunity to reduce the risk of virus-mediated insertions, which wouldimprove the safety of gene therapy, but also favors gene correction and vaccine development.

Codon Swapping of Zinc Finger Nucleases Confers Expression in Primary Cells and In Vivo from a Single Lentiviral Vector by Abarrategui-Pontes Cecilia, Creneguy Alison, Thinard Reynald, Fine Eli J., Thepenier Virginie, Fournier Le Ray Laure, Cradick Thomas J., Bao Gang, Tesson Laurent, Podevin Guillaume, Anegon Ignacio, Nguyen T. Huy (365-376).
Background: Zinc finger nucleases (ZFNs) are promising tools for genome editing for biotechnological as wellas therapeutic purposes. Delivery remains a major issue impeding targeted genome modification. Lentiviral vectors arehighly efficient for delivering transgenes into cell lines, primary cells and into organs, such as the liver. However, the reversetranscription of lentiviral vectors leads to recombination of homologous sequences, as found between and withinZFN monomers. Methods: We used a codon swapping strategy to both drastically disrupt sequence identity between ZFNmonomers and to reduce sequence repeats within a monomer sequence. We constructed lentiviral vectors encoding codonswappedZFNs or unmodified ZFNs from a single mRNA transcript. Cell lines, primary hepatocytes and newborn ratswere used to evaluate the efficacy of integrative-competent (ICLV) and integrative-deficient (IDLV) lentiviral vectors todeliver ZFNs into target cells. Results: We reduced total identity between ZFN monomers from 90.9% to 61.4% andshowed that a single ICLV allowed efficient expression of functional ZFNs targeting the rat UGT1A1 gene after codonswapping,leading to much higher ZFN activity in cell lines (up to 7-fold increase compared to unmodified ZFNs and 60%activity in C6 cells), as compared to plasmid transfection or a single ICLV encoding unmodified ZFN monomers. Offtargetanalysis located several active sites for the 5-finger UGT1A1-ZFNs. Furthermore, we reported for the first timesuccessful ZFN-induced targeted DNA double-strand breaks in primary cells (hepatocytes) and in vivo (liver) after deliveryof a single IDLV encoding two ZFNs. Conclusion: These results demonstrate that a codon-swapping approach alloweda single lentiviral vector to efficiently express ZFNs and should stimulate the use of this viral platform for ZFNmediatedgenome editing of primary cells, for both ex vivo or in vivo applications.

Reversal of Bone Cancer Pain by HSV-1-Mediated Silencing of CNTF in an Afferent Area of the Spinal Cord Associated with AKT-ERK Signal Inhibition by Xu Yang, Jia Liu, Zun-Jing Liu, Qing-Jie Xia, Mu He, Ran Liu, Wei Liu, Wei Wang, Jin Liu, Xin-Fu Zhou, Yun-Hui Zhang, Ting-Hua Wang (377-388).
Pain induced by bone metastases has a strong impact on the quality of life of patients with cancer, but currenttherapies for bone cancer pain cannot attain a satisfactory therapeutic goal because of various adverse reactions. Currently,advanced monitoring is required to clarify pathogenic mechanisms, so as to develop more effective treatments. We constructedherpes simplex virus carrying small interference RNA for CNTF (HSV-siCNTF) and established cancer-inducedbone cancer pain models with intra-tibial injection of MRMT-1 cells. At different time points after treatment, sensoryfunction indicated by thermal hyperalgesia and mechanical allodynia was measured. The mechanism underlying sensoryfunction regulated by CNTF was also determined. There was apparent mechanical and thermal hyperalgesia in rats injectedwith bone cancer cells. Bone destruction was detected in the area of tibia injected with tumor cells by the plain radiography.MRMT-1 cells and the increased number of osteoclasts were found in tibia sections stained with hematoxylinand eosin. Intrathecal injection of morphine or HSV-siCNTF significantly reduced the mechanical allodynia and thermalhyperalgesia, which was accompanied by astrocyte hypertrophy. The number of nerve fibers positive for substance P (SP)and calcitonin gene related peptide (CGRP) was significantly decreased, which was consistent with the decrease of CNTF,ERK/pERK, AKT/pAKT and c-fos expression. These results demonstrate that the HSV-siCNTF gene therapy appearsbeneficial for the treatment of pain induced by bone cancer via blocking the AKT-ERK signaling pathway. Our data suggestthat CNTF interference may be considered a new target to develop an effective management for bone cancer pain.

Identification of Vβ7.1_H3F7 as a Therapeutic Gene Encoding TCR Specific to Hepatocellular Carcinoma by Shulin Huang, Han Shen, Zhiming Li, Sung-Kay Chiu, Runsheng Ruan, Lanfeng Xiao, Chi-Meng Tzeng (389-399).
The feasibility of T-Cell receptor (TCR) gene therapy using a MART-1-specific TCR has been previouslydemonstrated in melanoma patients. However, it remains a challenge without a defined tumor-specific antigen in the therapyof hepatocellular carcinoma (HCC). In this study, through the analysis of clonal expansion of TCR Vβ subfamily andDNA sequencing, we identified TCR Vβ7.1_H3F7 as a potential therapeutic gene specifically for the HCC patients. Peripheralblood monouclear cells (PBMC) transfected with TCRV β7.1_H3F7 gene were specifically cytotoxic againstHCC cells in vitro. Adoptive transfer of this transfected PBMC resulted in a marked suppression of HCC tumor developmentin the animal model. These results demonstrated the value of TCRV β7.1_H3F7 as a therapeutic gene specificallyfor HCC. More importantly, it provides a novel strategy for screening tumor-specific TCR genes, which may pave theway for TCR gene therapy in cancer patients currently without the defined tumor-specific antigens.

SALL4: Engine of Cell Stemness by Jianhua Xiong (400-411).
The spalt (sal) family is a class of evolutionarily conserved genes originally identified in Drosophila as homeoticgenes required for embryonic development. In vertebrates, the expression of sal-like 4 (SALL4) is specifically enrichedin both embryonic and adult stem/stem-like cells. SALL4 is a master regulator that contributes to cell stemness in biologicaldevelopment and tumor growth. Thus, Sall4 has emerged as a target for gene therapy. In addition, numerous mutationsaffecting the Sall4 gene have been discovered and clinically linked to a series of congenital abnormalities, such asDuane/Duane-related syndromes, ventricular septal defect and premature ovarian failure. This review delineates the underlyingmechanisms of key functions of SALL4 and its use as a target for gene therapy. Finally, I summarize and discussadvances made on the application of Sall4 and its functions in diagnostics and treatments for human diseases.