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Viral vector-mediated reprogramming of the fibroblastic tumor stroma sustains curative melanoma treatment
The tumor microenvironment (TME) is a complex amalgam of tumor cells, immune cells, endothelial cells and fibroblastic stromal cells (FSC). Cancer-associated fibroblasts are generally seen as tumor-promoting entity. However, it is conceivable that particular FSC populations within the TME contribute to immune-mediated tumor control. Here, we show that intratumoral treatment of mice with a recombinant lymphocytic choriomeningitis virus-based vaccine vector expressing a melanocyte differentiation antigen resulted in T cell-dependent long-term control of melanomas.
Using single-cell RNA-seq analysis, we demonstrate that viral vector-mediated transduction reprogrammed and activated a Cxcl13-expressing FSC subset that show a pronounced immunostimulatory signature and increased expression of the inflammatory cytokine IL-33. Ablation of Il33 gene expression in Cxcl13-Cre-positive FSCs reduces the functionality of intratumoral T cells and unleashes tumor growth. Thus, reprogramming of FSCs by a self-antigen-expressing viral vector in the TME is critical for curative melanoma treatment by locally sustaining the activity of tumor-specific T cells..
Advances in the Development and the Applications of Non-viral, Episomal Vectors for Gene Therapy
Non-viral and non-integrating episomal vectors are reemerging as a valid, alternative technology to integrating viral vectors for gene therapy, due to their more favorable safety profile, significantly lower risk for insertional mutagenesis and a lesser potential for innate immune reactions, in addition to their low production cost
. Over the past few years, attempts have been made to generate highly functional non-viral vectors that display long-term maintenance within cells and promote more sustained gene expression relative to conventional plasmids. Extensive research into the parameters that stabilize the episomal DNA within dividing and non-dividing cells has shed light into the genetic and epigenetic mechanisms that govern replication and transcription of episomal DNA within a mammalian nucleus in long-term cell culture.
Episomal vectors based on scaffold/matrix attachment regions (S/MAR) do not integrate into the genomic DNA and address the serious problem of plasmid loss during mitosis by providing mitotic stability to established plasmids, which results in long-term transfection and transgene expression. The inclusion, in such vectors, of an origin of replication – IR – from the human genome has greatly enhanced their performance in primary cell culture
. A number of vectors that function as episomes have arisen, which are either devoid or depleted of harmful CpG sequences and bacterial genes, and their effectiveness, as well as that of non-integrating viral episomes, is enhanced when combined with S/MAR elements. As a result of these advances, a ‘S/MAR technology’ has emerged for the production of efficient episomal vectors. Significant research continues in this field and innovations, in combination with promising systems based on nanoparticles and potentially combined with physical delivery methods will enable the generation of optimized systems with scale-up and clinical application suitability utilizing episomal vectors.
Applications of Adeno-Associated Virus Vector-Mediated Gene Delivery for Neurodegenerative Diseases and Psychiatric Diseases: Progress, Advances, and Challenges
Neurodegeneration is the most common disease in the elderly population due to its slowly progressive nature of neuronal deterioration, eventually leading to executive dysfunction. The pathological markers of neurological disorders are relatively well-established, however, detailed molecular mechanisms of progression and therapeutic targets are needed to develop novel treatments in human patients.
Treating known therapeutic targets of neurological diseases has been aided by recent advancements in adeno-associated virus (AAV) technology. AAVs are known for their low-immunogenicity, blood-brain barrier (BBB) penetrating ability, selective neuronal tropism, stable transgene expression, and pleiotropy. In addition, the usage of AAVs has enormous potential to be optimized.
Therefore, AAV can be a powerful tool used to uncover the underlying pathophysiology of neurological disorders and to increase the success in human gene therapy. This review summarizes different optimization approaches of AAV vectors with their current applications in disease modeling, neural tracing and gene therapy, hence exploring progressive mechanisms of neurodegenerative diseases as well as effective therapy. Lastly, this review discusses the limitations and future perspectives of the AAV-mediated transgene delivery system.
Adenovirus type 5 vectors encoding short hairpin RNAs targeting dengue virus 5′ non-translated region and capsid gene suppress pre-established dengue infection in cultured epithelial and myeloid cells
Dengue, a mosquito-borne viral disease, caused by any of four serotypes of dengue viruses (DENV-1, -2, -3 and -4), is estimated to affect >1 million of the world’s population daily. We showed earlier that a recombinant human adenovirus type 5 (HuAd5) vector, encoding a short hairpin RNA (shRNA), targeting a conserved sequence in the DENV genome, could effectively suppress pre-established DENV-2 infection in Vero cells.
In this study, we identified an additional conserved shRNA target in the DENV genome, developed a HuAd5 vector to target this site, and evaluated if HuAd5-delivered shRNAs suppress pre-established infection by the remaining three DENV serotypes, not only in Vero cells, but also in macrophages, the in vivo sites of DENV replication in infected individuals. We also assessed the effect of anti-HuAd5 antibodies on shRNA delivery.
We show that recombinant HuAd5 vectors, encoding shRNAs targeting conserved DENV genomic sequences, in the 5′ non-translated region and capsid gene, can suppress ongoing replication of all four prototypic DENV serotypes in Vero cells and in a HuAd5-refractory human macrophage cell line expressing a DENV attachment factor. DENV suppression was assessed on the basis of inhibition of viral antigen secretion, viral RNA replication and progeny virus generation.
Interestingly HuAd5 vector-mediated DENV suppression in the macrophage cell line was dependent on the presence of anti-HuAd5 antibody. This suggests that HuAd5 vector complexed to its antibody enters these cells through the Fc receptor pathway. This may have implications for specific targeting of HuAd5 vector-mediated antiviral RNA interference therapy to macrophages.
An intelligent method based on feed-forward artificial neural network and least square support vector machine for the simultaneous spectrophotometric estimation of anti hepatitis C virus drugs in pharmaceutical formulation and biological fluid
This study proposed simple and reliable spectrophotometry method for simultaneous analysis of hepatitis C antiviral binary mixture containing sofosbuvir (SOF) and daclatasvir (DAC). This technique is based on the use of feed-forward artificial neural network (FF-ANN) and least square support vector machine (LS-SVM). FF-NN with Levenberg-Marquardt (LM) and Cartesian genetic programming (CGP) algorithms was trained to determine the best number of hidden layers and the number of neurons.
This comparison demonstrated that the LM algorithm had the minimum mean square error (MSE) for SOF (1.59 × 10-28) and DAC (4.71 × 10-28). In LS-SVM model, the optimum regularization parameter (γ) and width of the function (σ) were achieved with root mean square error (RMSE) of 0.9355 and 0.2641 for SOF and DAC, respectively. The coefficient of determination (R2) value of mixtures containing SOF and DAC was 0.996 and 0.997, respectively.
The percentage recovery values were in the range of 94.03-104.58 and 94.04-106.41 for SOF and DAC, respectively. Statistical test (ANOVA) was implemented to compare high-performance liquid chromatography (HPLC) and spectrophotometry, which showed no significant difference. These results indicate that the proposed method possesses great potential ability for prediction of concentration of components in pharmaceutical formulations.
