Current Gene Therapy - Volume 14, Issue 5, 2014

Triplex-forming peptide nucleic acids (PNAs) facilitate gene editing by stimulating recombination of donor DNAs 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 novel strategy where next generation single-stranded gamma PNAs (γPNAs) containing miniPEG substitutions at the gamma position can target genomic DNA in mouse bone marrow at mixed-sequence sites to induce targeted gene editing. In addition to 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 frequencies of 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 recognition without sequence restriction.

Suppression of tau protein expression has been shown to improve behavioral deficits in mouse models of tauopathies, offering an attractive therapeutic approach. Experimentally this had been achieved by switching off the promoters controlling the transgenic human tau gene (MAPT), which is not possible in human patients. The aim of the present study was therefore to evaluate the effectiveness of small interfering RNAs (siRNAs) and their cerebral delivery to suppress human tau expression in vivo, which might be a therapeutic option for human tauopathies. We used primary cortical neurons of transgenic mice expressing P301S-mutated human tau and Lund human mesencephalic (LUHMES) cells to validate the suppressive effect of siRNA in vitro. For measuring the effect in vivo, we stereotactically injected siRNA into the brains of P301S mice to reveal the suppression of tau by immunochemistry (AT180, MC1, and CP13 antibodies). We found that the Accell™ SMART pool siRNA against MAPT can effectively suppress tau expression in vitro and in vivo without 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 end labeling (TUNEL) assay showed neither signs of neurotoxicity or neuroinflammation nor apoptosis when MAPT siRNA is present in the hippocampus. Our data suggest that siRNA against MAPT can serve as a potential tool for gene therapy in tauopathies.

Lentiviruses are powerful tools for gene delivery and have been widely used for the dissection of gene functions in both replicating and quiescent cells. Recently, lentiviruses have also been used for delivering target sequences in gene therapy. Although the lentiviral system provides sustained exogenous gene expression, serious concerns have been raised due to its unfavorable insertion-mediated mutagenesis effect, thereby resulting in the silencing or activation of some unexpected genes. Thus, an array of modifications of the original vectors may reduce risks. Here, we briefly review the structure of the integrase protein, which is an essential protein for viral insertion and integration; the mechanisms of integrase-mediated integration; and the effects of the modifications of integrase. Moreover, we discuss the advantages resulting from integrase modifications and their future applications. Taken together, the generation of integrase-deficient lentivirus (IDLV) not only provides us with an opportunity to reduce the risk of virus-mediated insertions, which would improve the safety of gene therapy, but also favors gene correction and vaccine development.

Background: Zinc finger nucleases (ZFNs) are promising tools for genome editing for biotechnological as well as therapeutic purposes. Delivery remains a major issue impeding targeted genome modification. Lentiviral vectors are highly efficient for delivering transgenes into cell lines, primary cells and into organs, such as the liver. However, the reverse transcription of lentiviral vectors leads to recombination of homologous sequences, as found between and within ZFN monomers. Methods: We used a codon swapping strategy to both drastically disrupt sequence identity between ZFN monomers and to reduce sequence repeats within a monomer sequence. We constructed lentiviral vectors encoding codonswapped ZFNs or unmodified ZFNs from a single mRNA transcript. Cell lines, primary hepatocytes and newborn rats were used to evaluate the efficacy of integrative-competent (ICLV) and integrative-deficient (IDLV) lentiviral vectors to deliver ZFNs into target cells. Results: We reduced total identity between ZFN monomers from 90.9% to 61.4% and showed 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. Offtarget analysis located several active sites for the 5-finger UGT1A1-ZFNs. Furthermore, we reported for the first time successful ZFN-induced targeted DNA double-strand breaks in primary cells (hepatocytes) and in vivo (liver) after delivery of a single IDLV encoding two ZFNs. Conclusion: These results demonstrate that a codon-swapping approach allowed a single lentiviral vector to efficiently express ZFNs and should stimulate the use of this viral platform for ZFNmediated genome editing of primary cells, for both ex vivo or in vivo applications.

Pain induced by bone metastases has a strong impact on the quality of life of patients with cancer, but current therapies 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 constructed herpes simplex virus carrying small interference RNA for CNTF (HSV-siCNTF) and established cancer-induced bone cancer pain models with intra-tibial injection of MRMT-1 cells. At different time points after treatment, sensory function indicated by thermal hyperalgesia and mechanical allodynia was measured. The mechanism underlying sensory function regulated by CNTF was also determined. There was apparent mechanical and thermal hyperalgesia in rats injected with 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 hematoxylin and eosin. Intrathecal injection of morphine or HSV-siCNTF significantly reduced the mechanical allodynia and thermal hyperalgesia, 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 appears beneficial for the treatment of pain induced by bone cancer via blocking the AKT-ERK signaling pathway. Our data suggest that CNTF interference may be considered a new target to develop an effective management for bone cancer pain.

The feasibility of T-Cell receptor (TCR) gene therapy using a MART-1-specific TCR has been previously demonstrated in melanoma patients. However, it remains a challenge without a defined tumor-specific antigen in the therapy of hepatocellular carcinoma (HCC). In this study, through the analysis of clonal expansion of TCR Vβ subfamily and DNA sequencing, we identified TCR Vβ7.1_H3F7 as a potential therapeutic gene specifically for the HCC patients. Peripheral blood monouclear cells (PBMC) transfected with TCRV β7.1_H3F7 gene were specifically cytotoxic against HCC cells in vitro. Adoptive transfer of this transfected PBMC resulted in a marked suppression of HCC tumor development in the animal model. These results demonstrated the value of TCRV β7.1_H3F7 as a therapeutic gene specifically for HCC. More importantly, it provides a novel strategy for screening tumor-specific TCR genes, which may pave the way for TCR gene therapy in cancer patients currently without the defined tumor-specific antigens.

The spalt (sal) family is a class of evolutionarily conserved genes originally identified in Drosophila as homeotic genes required for embryonic development. In vertebrates, the expression of sal-like 4 (SALL4) is specifically enriched in both embryonic and adult stem/stem-like cells. SALL4 is a master regulator that contributes to cell stemness in biological development and tumor growth. Thus, Sall4 has emerged as a target for gene therapy. In addition, numerous mutations affecting the Sall4 gene have been discovered and clinically linked to a series of congenital abnormalities, such as Duane/Duane-related syndromes, ventricular septal defect and premature ovarian failure. This review delineates the underlying mechanisms of key functions of SALL4 and its use as a target for gene therapy. Finally, I summarize and discuss advances made on the application of Sall4 and its functions in diagnostics and treatments for human diseases.