Gene therapy prospects--intranasal delivery of therapeutic genes.
ABSTRACT Gene therapy is recognized to be a novel method for the treatment of various disorders. Gene therapy strategies involve gene manipulation on broad biological processes responsible for the spreading of diseases. Cancer, monogenic diseases, vascular and infectious diseases are the main targets of gene therapy. In order to obtain valuable experimental and clinical results, sufficient gene transfer methods are required. Therapeutic genes can be administered into target tissues via gene carriers commonly defined as vectors. The retroviral, adenoviral and adeno-associated virus based vectors are most frequently used in the clinic. So far, gene preparations may be administered directly into target organs or by intravenous, intramuscular, intratumor or intranasal injections. It is common knowledge that the number of gene therapy clinical trials has rapidly increased. However, some limitations such as transfection efficiency and stable and long-term gene expression are still not resolved. Consequently, great effort is focused on the evaluation of new strategies of gene delivery. There are many expectations associated with intranasal delivery of gene preparations for the treatment of diseases. Intranasal delivery of therapeutic genes is regarded as one of the most promising forms of pulmonary gene therapy research. Gene therapy based on inhalation of gene preparations offers an alternative way for the treatment of patients suffering from such lung diseases as cystic fibrosis, alpha-1-antitrypsin defect, or cancer. Experimental and first clinical trials based on plasmid vectors or recombinant viruses have revealed that gene preparations can effectively deliver therapeutic or marker genes to the cells of the respiratory tract. The noninvasive intranasal delivery of gene preparations or conventional drugs seems to be very encouraging, although basic scientific research still has to continue.
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ABSTRACT: The processes of lung fibrogenesis and fibrotic healing are common to a number of conditions with different etiologies. The lungs are the only affected organ in some cases, whereas in others, several organ systems are involved. Therapeutic options can be discussed from various perspectives. In this review, we address the localization of therapeutic targets with regard to cell compartments, including secreted ligands, cell surface, plasma membrane-cytosol interplay, cytosol and nucleus. Complex approach using stem cell therapy is also discussed. As the prognosis of patients with these disorders remains grim, treatment combinations targeting different molecules within the cell should sometimes be considered. It is reasonable to assume that blocking specific pathways will more likely lead to disease stabilization, while stem cell-based treatments could potentially restore lung architecture. Gene therapy could be a candidate for preventive care in families with proven specific gene polymorphisms and documented familial lung fibrosis. Chronobiology, that takes into account effect of circadian rhythm on cell biology, has demonstrated that timed drug administration can improve treatment outcomes. However, the specific recommendations for optimal approaches are still under debate. A multifaceted approach to interstitial lung disorders, including cooperation between those doing basic research and clinical doctors as well as tailoring research and treatment strategies toward (until now) unmet medical needs, could improve our understanding of the diseases and, above all, provide benefits for our patients.11/2014; 2(11):668-75. DOI:10.12998/wjcc.v2.i11.668
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ABSTRACT: The aim of the present study was to investigate the feasibility of lentiviral‑mediated Cys‑Asn‑Gly‑Arg‑Cys (CNGRC) peptide gene transduction in adipose stem cells. Adipose stem cells were prepared using enzymatic digestion and repeated adherence methods and identified in culture by immunofluorescence staining of surface markers. The pluripotency of the cultured adipose stem cells was confirmed by their induced differentiation into bone and fat cells. Following polymerase chain reaction amplification, the gene sequence for the CNGRC peptide was cloned into a lentiviral vector, which was then co‑transfected into 293T cells with packaging plasmids Helper 1.0 and Helper 2.0. The lentiviruses carrying the CNGRC peptide gene were then harvested and used to transfect adipose stem cells. Following transduction, expression of CNGRC in adipose stem cells was detected using western blot analysis. Adipose stem cells in culture were successfully induced to differentiate into adipocytes and osteoblasts and the lentiviral vector containing CNGRC‑3Flag‑EGFP was successfully constructed. Following transduction, western blot analysis and immunofluorescence staining demonstrated expression of the CNGRC protein in adipose stem cells. This suggested that adipose stem cell lines expressing the CNGRC peptide were successfully established.Molecular Medicine Reports 12/2014; DOI:10.3892/mmr.2014.3043 · 1.48 Impact Factor
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ABSTRACT: RNA interference technology is a widely used tool for the regulation of gene expression at the post-transcriptional level. One major challenge is to find the effective short interfering (si)RNA for target gene rapidly and easily, and then to deliver the siRNA into cells or tissues with high efficiency. Here, we designed a novel siRNA validation vector using a dual luciferase reporter system for the functional screening of effective RNAi targets in mammalian cells. Then, based on a siRNA expression cassette, we developed a derivative lentivirus delivery system to infect the appropriate cells or tissues for the efficient knockdown of target gene expression. Based on this system, we used human IRF7 gene, a key regulatory factor for the differentiation of monocytes to macrophages, as an example. We screened for the optimal siRNA, then packaged it into a lentiviral siRNA expression system. Then, monocytes were infected and we confirmed the knockdown of IRF7 expression could inhibit the differentiation of monocytes to macrophages. To validate our method further, we also screened and identified optimal siRNA for human TLR4 gene. In summary, we developed a novel siRNA validation system to identify optimal siRNA to target genes rapidly. In addition, the lentivirus system is an efficient tool for siRNA delivery for the further study of target gene function. Taken together, this represents an efficient and user-friendly strategy to validate and deliver siRNAs. Copyright © 2014. Published by Elsevier B.V.Gene 12/2014; 558(2). DOI:10.1016/j.gene.2014.12.063 · 2.20 Impact Factor