Development of hybrid viral vectors for gene therapy

Adenoviral, retroviral/lentiviral, adeno-associated viral, and herpesviral vectors are the major viral vectors used in gene therapy. Compared with non-viral methods, viruses are highly-evolved, natural delivery agents for genetic materials. Despite their remarkable transduction efficiency, both clinical trials and laboratory experiments have suggested that viral vectors have inherent shortcomings for gene therapy, including limited loading capacity, immunogenicity, genotoxicity, and failure to support long-term adequate transgenic expression. One of the key issues in viral gene therapy is the state of the delivered genetic material in transduced cells. To address genotoxicity and improve the therapeutic transgene expression profile, construction of hybrid vectors have recently been developed. By adding new abilities or replacing certain undesirable elements, novel hybrid viral vectors are expected to outperform their conventional counterparts with improved safety and enhanced therapeutic efficacy. This review provides a comprehensive summary of current achievements in hybrid viral vector development and their impact on the field of gene therapy.     

Molecular evolution of two duplicated CDPK genes CPK7 and CPK12 in grass species: a case study in wheat (Triticum aestivum L.)

Gene duplication contributes to the expansion of gene families and subsequent functional diversification. Calcium-dependent protein kinases (CDPKs) are members of an important calcium sensor family involved in abiotic and biotic stress signaling in plants. We report here the molecular evolution and expression analysis of a pair of duplicated CDPK genes CPK7 and CPK12 that arose in the common ancestor of grass species. With higher nonsynonymous/synonymous ratios (dN/dS, or ω), CPK12 genes appear to diverge more rapidly than CPK7s, suggesting relaxed selection constraints on CPK12s. Sliding window analysis revealed increased dN and ω values at N-terminal regions and the calcium-binding EF hand loops. Likelihood analyses using various models in PAML 4.0 showed purifying selection on both CPK7 and CPK12 lineages. In addition to the divergence in cis-element combinations on their promoters, functional divergence of CPK7 and CPK12 genes was also observed in wheat where TaCPK7 was found to respond to drought (PEG), salt (NaCl), cold, and hydrogen peroxide (H(2)O(2)) while TaCPK12 responded only to the treatment of ABA, a feature that may complement or expand TaCPK7-mediated stress signaling networks of wheat. The contrasting expression patterns of CPK7 and CPK12 genes under stress conditions were also observed in rice, suggesting conservative functional evolution of these genes. Since no positive selection was detected between the two lineages, the divergence of CPK7 and CPK12 genes should be ascribed to subfunctionalization, rather than neofunctionalization. Thus, our work demonstrates another case of evolutionary employment of duplicated genes via subfunctionalization for better adaptation.     

Reticulon-like proteins in Arabidopsis thaliana: structural organization and ER localization

Reticulons are proteins that have been found predominantly associated with the endoplasmic reticulum in yeast and mammalian cells. While their functions are still poorly understood, recent findings suggest that they participate in the shaping of the tubular endoplamic reticulum (ER). Although reticulon-like proteins have been identified in plants, very little is known about their cellular localization and functions. Here, we characterized the reticulon-like protein family of Arabidopsis thaliana. Three subfamilies can be distinguished on the basis of structural organization and sequence homology. We investigated the subcellular localization of two members of the largest subfamily, i.e. AtRTNLB2 and AtRTNLB4, using fluorescent protein tags. The results demonstrate for the first time that plant reticulon-like proteins are associated with the ER. Both AtRTNLB proteins are located in the tubular ER but AtRTNLB4 is also found in the lamellar ER cisternae, and in ER tubules in close association with the chloroplasts. Similarity in protein structure and subcellular localization between AtRTNLB2 and mammalian reticulons suggests that they could assume similar basic functions inside the cell.     

Differential contribution of the Fanconi anemia-related proteins to repair of several types of DNA damage in cultured silkworm cells

The silkworm Fanconi anemia (FA) pathway is required for normal cellular resistance to mitomycin C (MMC) in silkworms, but little is known about the requirement for repair of other types of DNA damage. Here we report that silkworm cells deficient for FA proteins FancD2 and FancM exhibit normal sensitivities to hydroxyurea (HU) and camptothecin (CPT), although FancM-dependent FancD2 monoubiquitination is induced upon these treatments. Similar results were observed in cells depleted for Rmi1 and Mhf1, which interact with the FancM protein. We also found that Rad51-knockdown cells exhibited normal sensitivity to HU despite induction of double-strand breaks by HU treatment.     

Chromatin Regulation of DNA Damage Repair and Genome Integrity in the Central Nervous System

With the continued extension of lifespan, aging and age-related diseases have become a major medical challenge to our society. Aging is accompanied by changes in multiple systems. Among these, the aging process in the central nervous system is critically important but very poorly understood. Neurons, as post-mitotic cells, are devoid of replicative associated aging processes, such as senescence and telomere shortening. However, because of the inability to self-replenish, neurons have to withstand challenge from numerous stressors over their lifetime. Many of these stressors can lead to damage of the neurons' DNA. When the accumulation of DNA damage exceeds a neuron's capacity for repair, or when there are deficiencies in DNA repair machinery, genome instability can manifest. The increased mutation load associated with genome instability can lead to neuronal dysfunction and ultimately to neuron degeneration. In this review, we first briefly introduce the sources and types of DNA damage and the relevant repair pathways in the nervous system (summarized in Fig. 1). We then discuss the chromatin regulation of these processes and summarize our understanding of the contribution of genomic instability to neurodegenerative diseases.     

Context-Dependent Remodeling of Rad51–DNA Complexes by Srs2 Is Mediated by a Specific Protein–Protein Interaction

The yeast Srs2 helicase removes Rad51 nucleoprotein filaments from single-stranded DNA (ssDNA), preventing DNA strand invasion and exchange by homologous recombination. This activity requires a physical interaction between Srs2 and Rad51, which stimulates ATP turnover in the Rad51 nucleoprotein filament and causes dissociation of Rad51 from ssDNA. Srs2 also possesses a DNA unwinding activity and here we show that assembly of more than one Srs2 molecule on the 3′ ssDNA overhang is required to initiate DNA unwinding. When Rad51 is bound on the double-stranded DNA, its interaction with Srs2 blocks the helicase (DNA unwinding) activity of Srs2. Thus, in different DNA contexts, the physical interaction of Rad51 with Srs2 can either stimulate or inhibit the remodeling functions of Srs2, providing a means for tailoring DNA strand exchange activities to enhance the fidelity of recombination.     

Decreased circulating miR-375: A potential biomarker for patients with non-small-cell lung cancer

MicroRNAs (miRNAs) are directly involved in cancer initiation, progression and metastasis. Alterations of miRNAs expression in cancer tissue may be reflected in circulation. We attempted to investigate the expression and clinical significance of plasma miR-20a, miR-31 and miR-375 in patients with non-small cell lung cancer (NSCLC). The plasma levels of miR-20a, miR-31 and miR-375 in 164 NSCLC patients and 164 healthy controls (discovery cohort) were evaluated and compared among various clinicopathological characteristics. The relationship between miRNA expression and clinical outcome of NSCLC patients was examined in an independent cohort (53 cases and 53 controls). The expression level of miR-375 in tissue was also examined. Plasma miR-375 levels in NSCLC patients were significantly decreased in both patient cohorts (P < 0.05). In addition, patients with metastatic NSCLC had lower plasma miR-375 expression than those with non-metastatic NSCLC (P < 0.05). Survival analysis showed that patients with low miR-375 expression had worse overall survival rates than those with high miR-375 expression (hazard ratios (HR) = 1.537 (1.046–2.258), P = 0.029). This association was independently validated in a separate cohort of 53 NSCLC patients (HR = 2.406, 95% CI 1.170–4.945, P = 0.017). The expression level of miR-375 was also found to be significantly down-regulated in NSCLC tissues compared with paracancerous tissues (P < 0.001). These findings indicate that miR-375 has an important role in NSCLC initiation and progression, and may be an independent poor prognostic factor in NSCLC patients.