Current Gene Therapy - Volume 22, Issue 1, 2022

Abstract: CRISPR (clustered regularly interspaced short palindromic repeats)/Cas gene editing is a revolutionary technology that can enable the correction of genetic mutations in vivo, providing great promise as a therapeutic intervention for inherited diseases. Adeno-associated viral (AAV) vectors are a potential vehicle for delivering CRISPR/Cas. However, they are restricted by their limited packaging capacity. Identifying smaller Cas orthologs that can be packaged, along with the required guide RNA elements, into a single AAV would be an important optimization for CRISPR/- Cas gene editing. Expanding the options of Cas proteins that can be delivered by a single AAV not only increases translational application but also expands the genetic sites that can be targeted for editing. This review considers the benefits and current scope of small Cas protein orthologs that are suitable for gene editing approaches using single AAV vector delivery.

Abstract: Lung adenocarcinoma (LUAD) is the common histological subtype of non-small-cell lung carcinoma (NSCLC). Circular RNAs (circRNAs) represent a new class of non-coding RNAs (ncRNAs) involved in the development of cancer. Accumulating evidence indicated that a large number of circular RNAs were found to be involved in many biological processes, including tumor initiation, proliferation and progression. These circRNAs present great potentials as new biomarkers and vital targets for disease diagnosis and prognosis. In this review, we mainly focus on the differentially expressed circRNAs and their functions in the pathogenesis of LUAD, which makes it possible for the utility of circRNAs as novel biomarkers for early diagnosis and therapy. Especially, it is helpful to develop circRNAs as crucial therapeutic targets, thus providing a promising biomedical application in the field of cancer gene therapy.

Abstract: Recently, genetic engineering by various strategies to stimulate gene expression in a specific and controllable mode is a speedily growing therapeutic approach. Genetic modification of human stem or progenitor cells, such as Embryonic Stem Cells (ESCs), Neural Progenitor Cells (NPCs), Mesenchymal Stem/Stromal Cells (MSCs), and Hematopoietic Stem Cells (HSCs) for direct delivery of specific therapeutic molecules or genes has been evidenced as an opportune plan in the context of regenerative medicine due to their supported viability, proliferative features, and metabolic qualities. On the other hand, a large number of studies have investigated the efficacy of modified stem cells in cancer therapy using cells from various sources, disparate transfection means for gene delivery, different transfected yields, and wide variability of tumor models. Accordingly, cell-based gene therapy holds substantial aptitude for the treatment of human malignancy as it could relieve signs or even cure cancer succeeding expression of therapeutic or suicide transgene products; however, there exist inconsistent results in this regard. Herein, we deliver a brief overview of stem cell potential to use in cancer therapy and regenerative medicine and importantly discuss stem cells based gene delivery competencies to stimulate tissue repair and replacement in concomitant with their potential to use as an anti-cancer therapeutic strategy, focusing on the last two decades’ in vivo studies.

Abstract: Hepatocellular carcinoma (HCC) is the sixth globally diagnosed cancer with a poor prognosis. Although the pathological factors of hepatocellular carcinoma are well elucidated, the underlying molecular mechanisms remain unclear. N⁶-methyladenosine (M⁶A) is adenosine methylation occurring at the N⁶ site, which is the most prevalent modification of eukaryotic mRNA. Recent studies have shown that M⁶A can regulate gene expression, thus modulating the processes of cell self-renewal, differentiation, and apoptosis. The methyls in M⁶A are installed by methyltransferases (“writers”), removed by demethylases (“erasers”) and recognized by M⁶A-binding proteins (“readers”). In this review, we discuss the roles of the above regulators in the progression and prognosis of HCC, and summarize the clinical association between M6A modification and hepatocellular carcinoma, so as to provide more valuable information for clinical treatment.

Background: Type 2 Diabetes Mellitus (T2DM) is a chronic disease. The molecular diagnosis should be helpful for the treatment of T2DM patients. With the development of sequencing technology, a large number of differentially expressed genes were identified from expression data. However, the method of machine learning can only identify the local optimal solution as the signature. Objective: The mutation information obtained by inheritance can better reflect the relationship between genes and diseases. Therefore, we need to integrate mutation information to more accurately identify the signature. Methods: To this end, we integrated Genome-Wide Association Study (GWAS) data and expression data, combined with expression Quantitative Trait Loci (eQTL) technology to get T2DM predictive signature (T2DMSig-10). Firstly, we used GWAS data to obtain a list of T2DM susceptible loci. Then, we used eQTL technology to obtain risk Single Nucleotide Polymorphisms (SNPs), and combined with the pancreatic β-cells gene expression data to obtain 10 protein-coding genes. Next, we combined these genes with equal weights. Results: After Receiver Operating Characteristic (ROC), single-gene removal and increase method, gene ontology function enrichment and protein-protein interaction network were used to verify the results showed that T2DMSig-10 had an excellent predictive effect on T2DM (AUC=0.99), and was highly robust. Conclusion: In short, we obtained the predictive signature of T2DM, and further verified it.

Background: Radiation-induced lung injury (RILI) is lacking effective therapeutic strategies. In this study, we conducted TGF-β1-based CRISPR/Cas9 gene therapy for RILI. Objective: Mouse lungs were irradiated with a single-dose of 20-Gy gamma rays followed by intravenous administration of Ad-CRISPR-TGF-β1 or Ad- CRISPR-Null. Methods: Haematoxylin and eosin staining, as well as Masson staining, were performed to observe lung morphology. Albumin and IgM concentrations in bronchoalveolar lavage fluid were measured by ELISA. Cytokine levels were measured using ELISA and/or real-time PCR with terminal deoxynucleotidyl transferase-mediated nick-end labelling. Results: Ad-CRISPR-TTGF-β1 improved histopathological and biochemical markers of lung injury, reduced secretion and expression of inflammatory cytokines, and inhibited progression of fibrosis. Importantly, the SK1/S1P axis, which is known to play a key role via S1P1 in TGF-β1-dependent S1PR pattern remodelling, is responsible for promoting fibrosis. Conclusion: Our results indicate novel insights for RILI therapy.

Aims: We aim to define parameters that affect the safety and long-term transgene expression of attenuated HSV-1 vectors and optimize the expression cassettes to achieve robust and sustained expression in CNS. Background: Engineered, attenuated Herpes Simplex Virus (HSV) vectors are promising vehicles for gene delivery to the peripheral and central nervous systems. The virus latent promoter (LAP) is commonly used to drive exogenous gene expression; however, parameters affecting the safety and longterm transgene expression of attenuated HSV-1 vectors have not been fully understood. Objective: The study aimed at using CRISPR-Cas9 system to construct attenuated HSV-1 vectors and examine the influence of transgene cassette construction and insertion site on transgene expression and vector safety. Methods: In this study, we used a CRISPR-Cas9 system to accurately and efficiently edit attenuated HSV-1 strain 1716, and construct two series of recombinant virus LMR and LMRx with different sets of gene cassettes insertion in Exon1(LAP2) and 2.0 kb intron downstream of LAP, respectively. The transgene expression and viral gene transcriptional kinetics were compared in in vitro cell lines. The reporter gene expression and safety profiles of each vector were further evaluated in mouse hippocampus gene transduction model. Results: The in vitro cell line analysis indicated that the insertion of a gene expression cassette would disrupt virus gene transcription. Mouse hippocampus transducing analysis suggested that complete expression cassette insertion at 2.0 kb intron could achieve robust and longtime gene expression than the other constructs. Recombinants with gene expression cassettes lacking Poly (A) induced significant neuronal inflammation due to persistent viral antigen expression and microglia activation. Conclusion: Our results indicated that the integrity of LAT transcripts was not necessary for establishment of long-term latent expression. Exogenous strong promoters (like cBh promoter) could remain active during latency when placed in Exon1 or 2.0 Kb Intron of LAT locus, although their transcriptional activity declined with time. Consistent with previous research, the foreign gene expression would last much longer when the gene cassette was located downstream of Exon1, which suggested a role of LAP2 in maintaining promoter activity during latency. Besides, over-transcription of the downstream part of LAT may induce continuous activation of the attenuated vectors, which suggests an important role of LAT in maintaining viral reactivation potential.