Gene Therapy of the Other Genome: The Challenges of Treating Mitochondrial DNA Defects

Human mitochondrial DNA is a 16.5 kb circular DNA molecule located inside the mitochondrial matrix. Although accounting for only about 1% of total cellular DNA, defects in mitochondrial DNA have been found to have major effects on human health. A single mtDNA mutation may cause a bewildering variety of clinical symptoms mainly involving the neuromuscular system at any age of onset. Despite significant advances in the understanding of mitochondrial DNA defects at a molecular level, the clinical diagnosis of mtDNA diseases remains a significant challenge and effective therapies for such diseases are as yet unavailable. In contrast to gene therapy for chromosomal DNA defects, mitochondrial gene therapy is a field that is still in its infancy and attempts towards gene therapy of the mitochondrial genome are rare. In this review we outline what we believe are the unique challenges associated with the correction of mtDNA mutations and summarize current approaches to gene therapy for the “other genome”.     

人轮状病毒基因组结构及功能研究进展

轮状病毒能够广泛、频繁地导致重症腹泻的发生,是目前世界范围内导致两岁以下婴幼儿严重腹泻病的首要病原体.近年来,有关人轮状病毒基因组结构及功能的研究倍受关注,本文对其作介绍.     

Expression of GFP in the Mitochondrial Compartment Using DQAsome-Mediated Delivery of an Artificial Mini-mitochondrial Genome

Purpose  

We describe a novel strategy for expression of GFP in mammalian mitochondria.     

Genomic Classifier for Patient Enrichment: Misclassification and Type I Error Issues in Pharmacogenomics Noninferiority Trial

Unlike the phenotypic characteristics, a genomic diagnostic assay is needed to assess whether a patient truly has the genomic characteristics of interest for therapeutic investigation. However, in reality, the diagnostic assay is not perfect without misclassification error. We consider a targeted or enrichment pharmacogenomics clinical trial with a noninferiority objective. We assume the probability of response of the misclassified subjects is identical in the treated and control groups because the blinding should yield no preferential misclassification. We show that the maximum Type I error rate associated with falsely concluding noninferiority can be substantially liberal if the accuracy of the diagnostic assay is much less than perfect. When the diagnostic assay has moderate to high sensitivity and specificity, a reasonable range of false nondisease prediction rate among test positives can be identified where the maximum Type I error rate inflation is much less. To achieve a prescribed level of positive predictive value, the feasibility of a genomic biomarker for enrichment in a noninferiority study is a function of disease prevalence. Given the evidential standard of much less inflation of Type I error rate can be shown, a genomic biomarker, if qualified, may be useful as an adjunct tool for patient selection.     

Emerging technologies from the Human Genome Project for understanding susceptibility and risk

The new technologies from the Human Genome Program provide exceptional opportunities for surveying and measuring human exposure, as well as determining susceptibility on an individual-by-individual basis. These new technologies will soon enable rapid screening of populations at risk, as well as the broader public, for a variety of genes known to be associated with increased risk. These include specific oncogenes, tumor suppressor genes and DNA repair enzymes. Use of these technologies also presents a number of ethical issues, both in screening and in use of the information about individuals. Overall, the use of rapid genotyping technologies will introduce a specificity and possible group identifiers that will present new challenges to the determination of risk within the EPA mandate.     

人类基因组拷贝数变异与新药研究开发

主要论述人类基因组拷贝数变异与新药研究开发的进展。参阅近年来文献,对基因拷贝数变异、基因拷贝数变异图谱与疾病关联的研究及重大疾病新药研究开发的进展进行综述。人类基因组拷贝数变异(CNVs)或称拷贝数多态性(CNPs)是人类基因组变异研究的新发展。首次综合性的人类基因组的拷贝数变异图谱为基因结构和人类疾病研究提供了一个重要的资源,为新药的研究开发开拓了更加广阔的前景。     

Mitochondrial Dynamics in SARS-COV2 Spike Protein Treated Human Microglia: Implications for Neuro-COVID

Journal of Neuroimmune Pharmacology - Emerging clinical data from the current COVID-19 pandemic suggests that ~ 40% of COVID-19 patients develop neurological symptoms attributed to...     

管忠震:临床试验对人类科学进步的贡献

药物临床试验这一领域我们国家对公众的宣传和教育不多，这是一个问题。特别是现在许多媒体一谈到临床试验其基本论调就是拿病人当白老鼠做实验。这样的舆论氛围导致了我们在招募病人的时候会遇到一些障碍，有些病人因为对科学的临床试验不了解而产生很多顾虑。     

人组织型纤溶酶原激活剂(t-PA)基因在烟草基因组中的整合

为了建立转基因植物生产t-PA的新方法，制备了整合有t-PA基因的转基因烟草植株。通过将t-PA cDNA从质粒pGEM-tPA中切下并插入含有CaMV35S及潮霉素筛选 标记的双元载体pCAMBIA1302中，构建成植物表达载体pCA1302NS-tPA，再将重组载体转入农杆菌GV3101中，通过叶盘法转化烟草。对筛选所得的转化植株进行Southern鉴定显示t-PA基因已整合到烟草基因组中。     

Human Dioxin Receptor Chimera Transactivation in a Yeast Model System and Studies on Receptor Agonists and Antagonists

Abstract: A yeast dioxin receptor chimera model has been developed to study ligand binding and transactivation properties of the human dioxin receptor. Using this new yeast model, the human dioxin receptor chimera was found to possess a constitutive transactivity on a LacZ reporter gene, however, the transactivation by the chimera was enhanced by the addition of several polycyclic aromatic hydrocarbons to the culture medium. The order of best polycyclic aromatic hydrocarbon inducer to worst correlated well with the known in vitro dioxin receptor binding affinities for these polycyclic aromatic hydrocarbons. 7, 8-Benzoflavone, a weak dioxin receptor agonist and strong antagonist of the mammalian dioxin receptor also behaved as a weak agonist and strong antagonist of the human dioxin receptor chimera expressed in yeast. The implications for these findings as well as the utility of this new yeast human dioxin receptor chimera model are discussed.     

Cloned GABA receptors are maintained in a stable cell line: allosteric and channel properties

The cloned cDNAs encoding the alpha 1 and beta 1 subunits of the bovine brain GABA(A) receptor have been co-transfected, using a dexamethasone-inducible promoter, into cultured hamster ovary cells, with selection to form a stable cell line. The use, alternatively, of a much stronger constitutive promoter led to cell death consequent upon high receptor density. After induction, the cells contained the alpha 1 and beta 1 mRNAs. The expressed receptors showed the high-affinity binding of [3H]muscimol and of the GABA(A) receptor channel blocker, t-butylphosphorothionate (TBPS), and the characteristic enhancement of the former by a pregnanolone. Their GABA-activated current was potentiated by the barbiturate, pentobarbitone, was reversibly blocked by bicuculline and picrotoxin, but was not enhanced by benzodiazepines. In mouse spinal cord neurons GABA activates channel openings to at least four conductance states (45, 30, 19 and 12 pS) with the 30 pS state being the most frequently observed (main) state. However, the main state of the alpha 1/beta 1 GABA(A) receptor was the 19 pS state. The enhancement of GABA(A) receptor current by barbiturates wa due to prolongation of mean channel lifetime, whereas the reduction of GABA(A) receptor current by picrotoxin was due to reduction of channel opening frequency and mean channel lifetime. Stable cell lines containing subunit combinations of this receptor should provide a powerful tool for the elucidation of its channel features and control mechanisms.     

Mitochondrial Delivery of Bongkrekic Acid Using a MITO-Porter Prevents the Induction of Apoptosis in Human HeLa Cells

The fact that mitochondrial dysfunction has been implicated in a variety of human diseases suggests that they would be expected as a target organelle for these diseases. Bongkrekic acid (BKA) is a chemical that functions as a ligand of the adenine nucleotide translocator and is known to potently inhibit the mitochondrial permeability transition that is associated with apoptosis. Thus, delivering it to mitochondria would be an innovative therapy for the treatment of mitochondrial diseases that are largely associated with apoptosis. Here, we report on the use of a MITO-Porter, an innovative nanocarrier for mitochondrial delivery via mitochondrial membrane fusion, for delivering BKA to mitochondria. We first constructed a BKA-MITO-Porter, in which BKA is contained in lipid envelopes of a MITO-Porter. We then confirmed that the BKA–MITO-Porter was efficiently internalized into cells and is delivered to mitochondria, similar to a conventional MITO-Porter. Moreover, we evaluated the antiapoptosis effect of the BKA–MITO-Porter in HeLa cells by measuring caspase 3/7 activity. The findings confirmed that the BKA–MITO-Porter showed a strong antiapoptosis effect compared with naked BKA. The results reported here demonstrate its potential for the use in therapies aimed at mitochondrial diseases, as a mitochondrial medicine candidate. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1008–1015, 2013