Current Gene Therapy - Volume 19, Issue 1, 2019

Background: As one of the main blinding ocular diseases, corneal blindness resulted from neovascularization that disrupts the angiogenic privilege of corneal avascularity. Following neovascularization, inflammatory cells are infiltrating into cornea to strengthen corneal injury. How to maintain corneal angiogenic privilege to treat corneal disease has been investigated for decades. Methodology: Local administration of viral and non-viral-mediated anti-angiogenic factors reduces angiogenic protein expression in situ with limited or free of off-target effects upon gene delivery. Recently, Mesenchymal Stem Cells (MSCs) have been studied to treat corneal diseases. Once MSCs are manipulated to express certain genes of interest, they could achieve superior therapeutic efficacy after transplantation. Discussion: In the text, we first introduce the pathological development of corneal disease in the aspects of neovascularization and inflammation. We summarize how MSCs become an ideal candidate in cell therapy for treating injured cornea, focusing on cell biology, property and features. We provide an updated review of gene-based therapies in animals and preclinical studies in the aspects of controlling target gene expression, safety and efficacy. Gene transfer vectors are potent to induce candidate protein expression. Delivered by vectors, MSCs are equipped with certain characters by expressing a protein of interest, which facilitates better for MSC-mediated therapeutic intervention for the treatment of corneal disease. Conclusion: As the core of this review, we discuss how MSCs could be engineered to be vector system to achieve enhanced therapeutic efficiency after injection.

Background: Myocardial infarction (MI) is the most severe ischemic heart disease and directly leads to heart failure till death. Target molecules have been identified in the event of MI including increasing angiogenesis, promoting cardiomyocyte survival, improving heart function and restraining inflammation and myocyte activation and subsequent fibrosis. All of which are substantial in cardiomyocyte protection and preservation of cardiac function. Methodology: To modulate target molecule expression, virus and non-virus-mediated gene transfer have been investigated. Despite successful in animal models of MI, virus-mediated gene transfer is hampered by poor targeting efficiency, low packaging capacity for large DNA sequences, immunogenicity induced by virus and random integration into the human genome. Discussion: Nanoparticles could be synthesized and equipped on purpose for large-scale production. They are relatively small in size and do not incorporate into the genome. They could carry DNA and drug within the same transfer. All of these properties make them an alternative strategy for gene transfer. In the review, we first introduce the pathological progression of MI. After concise discussion on the current status of virus-mediated gene therapy in treating MI, we overview the history and development of nanoparticle-based gene delivery system. We point out the limitations and future perspective in the field of nanoparticle vehicle. Conclusion: Ultimately, we hope that this review could help to better understand how far we are with nanoparticle-facilitated gene transfer strategy and what obstacles we need to solve for utilization of nanomedicine in the treatment of MI.

Introduction: Members of the adenosine deaminase acting on RNA (ADAR) family of enzymes consist of double-stranded RNA-binding domains (dsRBDs) and a deaminase domain (DD) that converts adenosine (A) into inosine (I), which acts as guanosine (G) during translation. Using the MS2 system, we engineered the DD of ADAR1 to direct it to a specific target. The aim of this work was to compare the deaminase activities of ADAR1-DD and various isoforms of ADAR2-DD. Materials and Methods: We measured the binding affinity of the artificial enzyme system on a Biacore ™ X100. ADARs usually target dsRNA, so we designed a guide RNA complementary to the target RNA, and then fused the guide sequence to the MS2 stem-loop. A mutated amber (TAG) stop codon at 58 amino acid (TGG) of EGFP was targeted. After transfection of these three factors into HEK 293 cells, we observed fluorescence signals of various intensities. Results: ADAR2-long without the Alu-cassette yielded a much higher fluorescence signal than ADAR2-long with the Alu-cassette. With another isoform, ADAR2-short, which is 81 bp shorter at the C-terminus, the fluorescence signal was undetectable. A single amino acid substitution of ADAR2-long-DD (E488Q) rendered the enzyme more active than the wild type. The results of fluorescence microscopy suggested that ADAR1-DD is more active than ADAR2-long-DD. Western blots and sequencing confirmed that ADAR1-DD was more active than any other DD. Conclusion: This study provides information that should facilitate the rational use of ADAR variants for genetic restoration and treatment of genetic diseases.

Introduction: Recent studies on CD19-specific chimeric antigen receptor (CAR)-modified T cells (CARTs) have demonstrated unprecedented successes in treating refractory and relapsed B cell malignancies. The key to the latest CART therapy advances can be attributed to the improved costimulatory signals in the CAR design. Methods: Here, we established several novel CARs by incorporating T cell signaling domains of CD28 in conjunction with intracellular signaling motif of 4-1BB, CD27, OX40, ICOS, and IL-15Rα. These novel CARs were functionally assessed based on a simple target cell killing assay. Results: The results showed that the CD28/IL-15Rα co-signaling (153z) CAR demonstrated the fastest T cell expansion potential and cytotoxic activities. IL-15 is a key cytokine that mediates immune effector activities. The 153z CARTs maintained prolonged killing activities after repetitive rounds of target cell engagement. Consistent with the enhanced target killing function, the 153z CARTs produced increased amount of effector cytokines including IFN-γ, TNFα and IL-2 upon interaction with the target cells. Conclusion: In a follow-up clinical study, an acute lymphoblastic leukemia (ALL) patient, who experienced multiple relapses of central nervous system leukemia (CNSL) and failed all conventional therapies, was enrolled to receive the CD19-specific 153z CART treatment. The patient achieved complete remission after the 153z CART cell infusion. The translational outcome supports further investigation into the safety and enhanced therapeutic efficacy of the IL-15Rα-modified CART cells in cancer patients.

Background: Both Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) suicide gene therapy and exogenous CD40 ligand (CD40L)-CD40 interaction in cancer via conditionally replicating adenovirus can selectively kill tumors without damaging normal tissues. Objective: To further improve the cancer killing effect, we investigated the therapeutic effect of combined cancer gene therapy based on a selective oncolytic adenovirus vector containing Dm-dNK suicide gene and exogenous CD40L on breast carcinoma cells in vitro and in vivo. Methods: A series of conditionally replicating adenoviruses using adenovirus vector P74 were generated: P74-dNK, P74-CD40L (expressing Dm-dNK or CD40L respectively), and P74-dNK-CD40L (expressing combined Dm-dNK and CD40L). Breast cancer cell lines (MDA-MB-231, MCF-7) and non-tumor cell line (MRC5) were treated with adenovirus and cytotoxicity determined by MTT assay, and apoptosis assessed by flow cytometry after 72h. We also assessed in vivo cell killing efficiency using a mouse xenograft model with MDA-MB-231 cells. Results and Discussion: Co-expression of Dm-dNK and CD40L reduced cell proliferation of MDAMB- 231 or MCF7 cancer cells, and induced more apoptosis in TERT and CD40 positive cancer cells, but not normal MRC5 cells. Significant reduction in tumor volume was also seen in combined treatment arms as compared to any single treatment. Conclusion: Our data suggest enhanced, selective tumor cell killing using combined gene therapy with conditionally replicating adenovirus containing Dm-dNK suicide gene and exogenous CD40 ligation (CD40L-CD40).