Overexpression of multidrug resistance-associated protein 1 protects against cardiotoxicity by augmenting the doxorubicin efflux from cardiomyocytes

Doxorubicin is a commonly used anti-cancer drug used in treating a variety of malignancies. However, a major adverse effect is cardiotoxicity, which is dose dependent and can be either acute or chronic. Doxorubicin causes injury by DNA damage, the formation of free reactive oxygen radicals and induction of apoptosis. Our aim is to induce expression of the multidrug resistance-associated protein 1 (MRP1) in cardiomyocytes derived from human iPS cells (hiPSC-CM), to determine whether this will allow cells to effectively remove doxorubicin and confer cardioprotection. We generated a lentivirus vector encoding MRP1 (LV.MRP1) and validated its function in HEK293T cells and stem cell-derived cardiomyocytes (hiPSC-CM) by quantitative PCR and western blot analysis. The activity of the overexpressed MRP1 was also tested, by quantifying the amount of fluorescent dye exported from the cell by the transporter. We demonstrated reduced dye sequestration in cells overexpressing MRP1. Finally, we demonstrated that hiPSC-CM transduced with LV.MRP1 were protected against doxorubicin injury. In conclusion, we have shown that we can successfully overexpress MRP1 protein in hiPSC-CM, with functional transporter activity leading to protection against doxorubicin-induced toxicity.     

Surface modification of lipid nanoparticles for gene therapy

Gene therapies have the potential to target and effectively treat a variety of diseases including cancer as well as genetic, neurological, and autoimmune disorders. Although we have made significant advances in identifying non-viral strategies to deliver genetic cargo, certain limitations remain. In general, gene delivery is challenging for several reasons including the instabilities of nucleic acids to enzymatic and chemical degradation and the presence of restrictive biological barriers such as cell, endosomal and nuclear membranes. The emergence of lipid nanoparticles (LNPs) helped overcome many of these challenges. Despite its success, further optimization is required for LNPs to yield efficient gene delivery to extrahepatic tissues, as LNPs favor accumulation in the liver after systemic administration. In this mini-review, we provide an overview of current preclinical approaches in that LNP surface modification was leveraged for cell and tissue targeting by conjugating aptamers, antibodies, and peptides among others. In addition to their cell uptake and efficiency-enhancing effects, we outline the (dis-)advantages of the different targeting moieties and commonly used conjugation strategies.     

Extracellular matrix dynamics in heart failure: A prospect for gene therapy

In chronic congestive heart failure, an illness affecting more than 4 million Americans, there is impairment of myocardial extracellular matrix (ECM) remodeling. Failing human ventricular myocardium contains activated matrix metalloproteinases (MMPs), which are involved in adverse ECM remodeling. Our studies support the concept that impaired ECM remodeling and MMP activation are, in part, responsible for the cardiac structural deformation and heart failure. There is no known program that has declared its aim the investigation of the role of ECM gene therapy in heart failure. The development of transgenic technology, and emerging techniques for in vivo gene transfer, suggest a strategy for improving cardiac function by overexpressing or downregulation of the ECM components such as MMPs, tissue inhibitor of metalloproteinases (TIMPs), transforming growth factor-β1 (TGF-β), decorin, and collagen in cardiomyopathy and heart failure. J. Cell. Biochem. 68:403–410, 1998. © 1998 Wiley-Liss, Inc.     

Transplants of Human Mesenchymal Stem Cells Improve Functional Recovery After Spinal Cord Injury in the Rat

Human mesenchymal stem cells (hMSCs) derived from adult bone marrow represent a potentially useful source of cells for cell replacement therapy after nervous tissue damage. They can be expanded in culture and reintroduced into patients as autografts or allografts with unique immunologic properties. The aim of the present study was to investigate (i) survival, migration, differentiation properties of hMSCs transplanted into non-immunosuppressed rats after spinal cord injury (SCI) and (ii) impact of hMSC transplantation on functional recovery. Seven days after SCI, rats received i.v. injection of hMSCs (2×10⁶ in 0.5 mL DMEM) isolated from adult healthy donors. Functional recovery was assessed by Basso–Beattie–Bresnahan (BBB) score weekly for 28 days. Our results showed gradual improvement of locomotor function in transplanted rats with statistically significant differences at 21 and 28 days. Immunocytochemical analysis using human nuclei (NUMA) and BrdU antibodies confirmed survival and migration of hMSCs into the injury site. Transplanted cells were found to infiltrate mainly into the ventrolateral white matter tracts, spreading also to adjacent segments located rostro-caudaly to the injury epicenter. In double-stained preparations, hMSCs were found to differentiate into oligodendrocytes (APC), but not into cells expressing neuronal markers (NeuN). Accumulation of GAP-43 regrowing axons within damaged white matter tracts after transplantation was observed. Our findings indicate that hMSCs may facilitate recovery from spinal cord injury by remyelinating spared white matter tracts and/or by enhancing axonal growth. In addition, low immunogenicity of hMSCs was confirmed by survival of donor cells without immunosuppressive treatment.     

Neuroglobin基因体内转染对水杨酸钠给药后小鼠下丘核神经元听反应的影响

实验以48只成年健康昆明小鼠为实验对象,研究GeneJamer转染试剂介导的neuroglobin(NGB)基因体内转染对水杨酸钠给药后小鼠下丘核区听反应的影响。实验分4组,每组12只。A1组:对照组1(阴性对照,将GeneJamer转染试剂6μl和pEGFP-C12μg混合后注入下丘核脑区);A2组:对照组2[阳性对照,将GeneJamer转染试剂(6μl)和pEGFP-NGB(质粒载体pEGFP-C1与NGB基因全编码序列构建的重组子2μg)混合后注入下丘核脑区];B组:水杨酸钠给药组(450mg/kg·d-1)+pEGFP-C1;C组:水杨酸钠(450mg/kg.d-1)+pEGFP-NGB组。以直接注射法将GeneJamer转染试剂和重组质粒pEGFP-NGB混合后注入小鼠下丘核区。采用RT-PCR和Westernblot技术检测小鼠下丘核区NGBmRNA和蛋白的表达;采用细胞外记录技术,研究小鼠下丘核区神经元在水杨酸钠给药后转染重组质粒pEGFP-NGB对强度-发放率函数(刺激声强与实验鼠下丘核区神经元在接受声刺激所产生的电发放的关系曲线)、强度-潜伏期函数(刺激声强与实验鼠下丘核区神经元在接受声刺激至产生电发放潜伏期之间的关系曲线)和频率调谐曲线(实验鼠下丘核区神经元在各个频率纯音刺激下起反应的阈值绘制的曲线)的影响。实验观察到:(1)经GeneJamer转染试剂介导NGB基因可有效地转染小鼠下丘核区脑组织并得到表达。(2)水杨酸钠给药后神经元的强度-发放率函数曲线升高。对照组A1、A2各项指标进行比较均无统计学意义。对照组A1、A2和水杨酸钠+pEGFP-NGB组神经元的强度-发放率函数以非单调型(随刺激强度增加时,发放率表现为先降后升呈“V”形或“U”形)为主,分别占74.6%、72.2%和59.3%,水杨酸钠给药组以不规则型强度-发放率函数为主,占47%,与对照组A1、A2和水杨酸钠+pEGFP-NGB组比较,有显著性差异(P<0.01、P<0.01、P<0.05)。(3)水杨酸钠给药后神经元的强度-潜伏期函数曲线降低。对照组A1、A2各项指标进行比较均无统计学意义。水杨酸钠给药组以非单调型强度-潜伏期率函数为主,与对照组A1、A2和水杨酸钠+pEGFP-NGB组比较,有显著性差异(P<0.01、P<0.05)。(4)A1和A2对照组听反应神经元的调谐曲线,Q-10dB值均大于5.00,其调谐曲线为狭窄型。记录水杨酸钠给药组72个听神经元的调谐曲线,有53个神经元的Q-10dB值小于5.00,Q-10dB值最小为2.12,其调谐曲线为宽阔型;其余19个神经元的Q-10dB值大于5.00,属于狭窄型调谐曲线。水杨酸钠+pEGFP-NGB组67个听神经元的调谐曲线,有12个神经元的Q-10dB值小于5.00,Q-10dB值最小为2.87,其调谐曲线为宽阔型,其它的神经元的值大于5.00。它们的调谐曲线均属狭窄型。以上结果提示外源性NGB基因在水杨酸钠给药后小鼠下丘核区局部高表达,提示GeneJamer转染试剂介导NGB体内转基因治疗水杨酸钠引起的下丘核区的损伤的方法是可行的。实验小鼠转染NGB基因后可逆转因水杨酸钠给药引起的强度-发放率函数曲线升高以及强度-潜伏期函数曲线降低,并可逆转水杨酸钠引起的部分听神经元对声刺激强度的编码类型。     

Aptamer‐conjugated mesoporous polydopamine for docetaxel targeted delivery and synergistic photothermal therapy of prostate cancer

ObjectivesIt is imperative to develop efficient strategies on the treatment of prostate cancer. Here, we constructed multifunctional nanoparticles, namely AS1411@MPDA‐DTX (AMD) for targeted and synergistic chemotherapy/photothermal therapy of prostate cancer.Materials and MethodsMesoporous polydopamine (MPDA) nanoparticles were prepared by a one‐pot synthesis method, DTX was loaded through incubation, and AS1411 aptamer was modified onto MPDA by the covalent reaction. The prepared nanoparticles were characterized by ultra‐micro spectrophotometer, Fourier transform infrared spectra, transmission electron microscope, and so on. The targeting ability was detected by selective uptake and cell killing. The mechanism of AMD‐mediated synergistic therapy was detected by Western blot and immunofluorescence.ResultsThe prepared nanoparticles can be easily synthesized and possessed excellent water solubility, stability, and controlled drug release ability, preferentially in acidic context. Based on in vitro and in vivo results, the nanoparticles can efficiently target prostate cancer cells, promote DTX internalization, and enhance the antitumor effects of chemo‐photothermal therapy strategies under the NIR laser irradiation.ConclusionsAs a multifunctional nanoplatform, AS1411@MPDA‐DTX could efficiently target prostate cancer cells, promote DTX internalization, and synergistically enhance the antiprostate cancer efficiency by combining with NIR irradiation.     

基因治疗与人类健康

随着分子生物学及基因工程技术的迅猛发展 ,基因治疗已经成为治疗人类疾病的重要方法之一 ,同时也是维护人类健康最有发展前景的手段之一。诸如遗传病、肿瘤、和传染病与心血管病的基因治疗。遗传免疫方面 ,病毒性疾病和肿瘤的基因治疗 ,如将病毒抗原基因 (HBsAg)及一些肿瘤抗原基因 (CEA)直接注入人体内而产生抗体 ;人类亚健康状态 ,如肥胖、秃顶、疲劳、衰老等的基因治疗。然而基因治疗目前仍面临着许多困扰 ,如基因治疗的有效性、安全性、及社会伦理等诸多问题 ,因此在临床实际应用中要慎之又慎。只有对基因治疗合理规范和正确引导并遵循伦理原则 ,才能最终推动现代医学的发展。     

Filtroporation: A simple, reliable technique for transfection and macromolecular loading of cells in suspension.

Cultured Chinese hamster ovary (CHO) cells suspended in their growth medium were forced by gas pressure through the uniformly sized micropores of filter membranes. This procedure caused transient damage to the plasma membrane, which increased the permeability of the cells to exogenous molecules. This "filtroporation" was indicated by uptake of fluorescent dextran molecules up to 500,000 MW in cells deemed viable by trypan blue dye exclusion. The macromolecular uptake was increased if the driving pressure was increased at constant micropore size, or if the micropore size was decreased at constant driving pressure. Larger membrane perturbations permitted uptake of a luciferase reporter plasmid, which resulted in transfection of the CHO cells with the surviving cells expressing luciferase activity after 2 days in culture. This simple and general new method of porating cells in suspension may be optimized to incorporate the desired macromolecules while retaining the maximum viability.     

Transduction of modified factor VIII gene improves lentiviral gene therapy efficacy for hemophilia A

Hemophilia A (HA) is a bleeding disorder caused by deficiency of the coagulation factor VIII (F8). F8 replacement is standard of care, whereas gene therapy (F8 gene) for HA is an attractive investigational approach. However, the large size of the F8 gene and the immunogenicity of the product present challenges in development of the F8 gene therapy. To resolve these problems, we synthesized a shortened F8 gene (F8-BDD) and cloned it into a lentiviral vector (LV). The F8-BDD produced mainly short cleaved inactive products in LV-transduced cells. To improve F8 functionality, we designed two novel F8-BDD genes, one with an insertion of eight specific N-glycosylation sites (F8-N8) and another which restored all N-glycosylation sites (F8-299) in the B domain. Although the overall protein expression was reduced, high coagulation activity (>100-fold) was detected in the supernatants of LV-F8-N8- and LV-F8-299-transduced cells. Protein analysis of F8 and the procoagulation cofactor, von Willebrand Factor, showed enhanced interaction after restoration of B domain glycosylation using F8-299. HA mouse hematopoietic stem cell transplantation studies illustrated that the bleeding phenotype was corrected after LV-F8-N8 or -299 gene transfer into the hematopoietic stem cells. Importantly, the F8-299 modification markedly reduced immunogenicity of the F8 protein in these HA mice. In conclusion, the modified F8-299 gene could be efficiently packaged into LV and, although with reduced expression, produced highly stable and functional F8 protein that corrected the bleeding phenotype without inhibitory immunogenicity. We anticipate that these results will be beneficial in the development of gene therapies against HA.     

Low-dose whole thorax radiation therapy for COVID-19 pneumonia: inpatient onboarding process for a randomized controlled trial

BackgroundThe mortality of the SARS-CoV-2 virus (COVID-19) has been associated with a pulmonary inflammatory response resulting in hypoxemia and rapid clinical decline. PREVENT is an ongoing prospective multicenter Phase II randomized controlled trial where patients hospitalized with COVID-19 pneumonia are randomized to low dose radiation therapy (RT) versus control (clinicaltrials.gov, {"type":"clinical-trial","attrs":{"text":"NCT04466683","term_id":"NCT04466683"}}NCT04466683). We describe the inpatient onboarding process of the center contributing the largest number of patients to this trial.Materials and methodsCOVID-19 hospital admissions were attained by the clinical research manager and radiation oncologist daily. Text message contact was made with infectious disease, critical care, and nursing staff with reciprocal discussion of the trial protocol and approval for virtual consulting of the patient. Witnessed informed consent was obtained first by telephone and later in person. Simulation and treatment (performed without a computer plan) was performed on a linear accelerator with one personal protective equipment-protected therapist moving in and out of the treatment room, and a second therapist manning the console. Following on-site dose calculation by physics, the radiation oncologist approved the fields prior to treatment delivery.ResultsBetween August 28, 2020 and October 6, 2020, the first 10 enrolled patients on this multicenter trial were randomized and treated at our institution; no team member (research staff, radiation oncology) contracted COVID-19 while employing this protocol.ConclusionThis represents the first published protocol to address efficient and safe recruitment of COVID-19 patients for a radiation oncology trial, serving as a model for conducting recruitment of COVID-19 patients for clinical trials.