Mucus-penetrating nonviral gene vaccine processed in the epithelium for inducing advanced vaginal mucosal immune responses

Establishment of vaginal immune defenses at the mucosal interface layer through gene vaccines promise to prevent infectious diseases among females.Mucosal barriers composed of a flowing mucus hydrogel and tightly conjugated epithelial cells(ECs),which represent the main technical difficulties for vaccine development,reside in the harsh,acidic human vaginal environment.Different from frequently employed viral vectors,two types of nonviral nanocarriers were designed to concurrently overcome the barriers and induce immune responses.Differing design concepts include the charge-reversal property(DRLS)to mimic a virus that uses any cells as factories,as well as the addition of a hyaluronic acid coating(HA/RLS)to directly target dendritic cells(DCs).With a suitable size and electrostatic neutrality,these two nanoparticles penetrate a mucus hydrogel with similar diffusivity.The DRLS system expressed a higher level of the carried human papillomavirus type 16 L1 gene compared to HA/RLS in vivo.Therefore it induced more robust mucosal,cellular,and humoral immune responses.Moreover,the DLRS applied to intravaginal immunization induced high IgA levels compared with intramuscularly injected DNA(naked),indicating timely protection against pathogens at the mucus layer.These findings also offer important approaches for the design and fabrication of nonviral gene vaccines in other mucosal systems.

WHAT CAN WE LEARN FROM VIRUS IN DESIGNING NONVIRAL GENE VECTORS

Gene therapy has emerged as a potential new approach to treat genetic disorders by delivering therapeutic genes to target diseased tissues. However, its clinical use has been impeded by gene delivery systems. The viral vectors are very efficient in delivering and expressing their carried genes, but they have safety issues in clinical use. While nonviral vectors are much safer with very low risks after careful material design, but their gene transcription efficiency is too low to be clinically used. Thus, rational design of nonviral vectors mimicking the viral vectors would be a way to break this bottleneck. This review compares side-by-side how viral/nonviral gene vectors transcend these biological barriers in terms of blood circulation, cellular uptake, endosome escape, nucleus import and gene transcription.

Small interfering RNAs based therapies for intracerebral hemorrhage: challenges and progress in drug delivery systems

Intracerebral hemorrhage(ICH)is a subtype of stroke associated with higher rates of mortality.Currently,no effective drug treatment is available for ICH.The molecular pathways following ICH are complicated and diverse.Nucleic acid therapeutics such as gene knockdown by small interfering RNAs(siRNAs)have been developed in recent years to modulate ICH’s destructive pathways and mitigate its outcomes.However,siRNAs delivery to the central nervous system is challenging and faces many roadblocks.Existing barriers to systemic delivery of siRNA limit the use of naked siRNA;therefore,siRNA-vectors developed to protect and deliver these therapies into the specific-target areas of the brain,or cell types seem quite promising.Efficient delivery of siRNA via nanoparticles emerged as a viable and effective alternative therapeutic tool for central nervous system-related diseases.This review discusses the obstacles to siRNA delivery,including the advantages and disadvantages of viral and nonviral vectors.Additionally,we provide a comprehensive overview of recent progress in nanotherapeutics areas,primarily focusing on the delivery system of siRNA for ICH treatment.

Expression and Activities Experiment of DNA Transduction Motif Based on GAL4 in Pichia Pastoris

The genes encoding DNA-binding domain（BD） designed based on the yeast transcriptional activator GAL4 and protein transduction domain of HIV-1 Tat protein were fused via soft linker peptide sequence, and cloned into yeast expression vector pPIC9k. The resulted plasmid pTG was linearized and transfected into Pichia pastoris strains GS 115 by electroporation. High copies of transformants were obtained with Muts and HIS＋ phenotype identi- fication, PCR amplification and screening of G418. After flask culture and expression induced by methanol, the target protein named TG was well expressed and analyzed by SDS-PAGE and Western blot. Under optimized conditions, the yield of soluble recombinant protein was approximately 39.7 mg/L. DNA binding activity and cell transduction property of TG were analyzed by gel eleetrophoresis and fluorescent microscopy. The results show that the recombinant protein could bind strongly to the plasmid containing upstream activating sequence（UAS）. The cell experiments revealed that TG could deliver the binding plasmid into HEK-293 cells effectively. In summary, the work presented here suggests that TG is specific toward UAS containing plasmid and has the potential for use as nonviral DNA delivery agent.

MSC based gene delivery methods and strategies improve the therapeutic efficacy of neurological diseases

Mesenchymal stem cells(MSCs)are promising seed cells for neural regeneration therapy owing to their plasticity and accessibility.They possess several inherent characteristics advantageous for the transplantation-based treatment of neurological disorders,including neural differentiation,immunosuppression,neurotrophy,and safety.However,the therapeutic efficacy of MSCs alone remains unsatisfactory in most cases.To improve some of their abilities,many studies have employed genetic engineering to transfer key genes into MSCs.Both viral and nonviral methods can be used to overexpress therapeutic proteins that complement the inherent properties.However,to date,different modes of gene transfer have specific drawbacks and advantages.In addition,MSCs can be functionalized through targeted gene modification to facilitate neural repair by promoting neural differentiation,enhancing neurotrophic and neuroprotective functions,and increasing survival and homing abilities.The methods of gene transfer and selection of delivered genes still need to be optimized for improved therapeutic and targeting efficacies while minimizing the loss of MSC function.In this review,we focus on gene transport technologies for engineering MSCs and the application of strategies for selecting optimal delivery genes.Further,we describe the prospects and challenges of their application in animal models of different neurological lesions to broaden treatment alternatives for neurological diseases.

Transient expression of hepatitis B virus surface antigen (HBsAg) and human erythropoietin (hEPO) genes in milk after direct introduction into ewe

The ability to deliver nonviral DNA directly into animals would provide major practical and economic benefits. To pursue this potential, as a preliminary approach, the expression in mammary epithelial cells with nonviral DNA encoding HBsAg and hEPO was investigated by in vivo delivery directly into mammary gland. Either the bovine αs1 casein HBsAg or the WAP hEPO gene construct was introduced into mammary glands of dose by mammary artery injection. A single injection of 10 11 copies of DNA construct was implemented for a doe. Transient expression of the constructs in milk collected from milk letdown by induction up to 19 d after injection was measured with an hEPO ELISA kit from Boehringer Mannheim (Cat. No.1 693 417) for rhEPO, and a HBsAg ELISA kit (Abbott Lab, USA) for rHBsAg. The treated animals showed mean concentration of recombinant molecules at 16 μg/L for rHBsAg, and 2 mg/L for rhEPO in milk.

Antitumor and off-target effects of cholesterol-conjugated let-7a mimics in an orthotopic hepatocellular carcinoma xenograft nude mouse model

Objective: To explore the antitumor and potential off-target effects of systemically delivered cholesterol-conjugatedlet-7a mimics(Chol-let-7a) and control mimics(Chol-miRCtrl) on hepatocellular carcinomain vivo.Methods: The antitumor effects of two intravenous dosing regimens ofChol-let-7a on heptocellular carcinoma growth were compared using an orthotopic xenograft mouse model. Off-targets were analyzed with histopathological and ultrapathological features of heparenal tissue and cells in theChol-let-7a-, Chol-miRCtrl-, and saline-treated (blank) xenograft mice and normal control mice. Then,let-7a abundance in orthotopic tumors, corresponding paracancerous hepatic tissue, and normal liver tissue from healthy nude mice was examined by reverse transcription-polymerase chain reaction. The distribution ofChol-let-7a andChol-miRCtrl in vivo was examined by whole-animal imaging and frozen-sections observation. The experiments were approved by the Institutional Research Board of Peking Union Medical College Hospital.Results: Continuous treatment withChol-let-7a resulted in tumors that were 35.86% and 40.02% the size of those in theChol-miRCtrl and blank xenograft group (P < 0.01 andP < 0.01, respectively), while intermittent dosing withChol-let-7a resulted in tumors that were 65.42% and 56.66% the size of those in theChol-miRCtrl and the blank control group, respectively (P < 0.05 andP < 0.05). In addition, some histopathological and ultrapathological features were only observed after treatment with the two cholesterol-conjugated molecules, however mild with intermittent dosingChol-let-7a treatment, such as diffuse sinusoidal dilation and edema, primarily around the centrolobular vein in heptic tissues;mild hypercellularity with dilated capillary lumens in the renal tissue;and some organelle abnormalities found in heptic and renal cells. Furthermore, whole-animal imaging showed thatChol-let-7a andChol-miRCtrl were predominantly distributed in the liver, kidney, and bladder regions after injection, and that the concentration ofChol-let-7a andChol-miRCtrl in the kidney and the bladder decreased much slowly in the xenograft animals, especially in theChol-miRCtrl group. Finally, RT-PCR analysis showed thatlet-7a levels were significantly increased in Chol-let-7a-treated xenografts compared withChol-miRCtrl group (P=0.003) and blank xenograft group (P=0.001);however, the level was only equivalent to 50.6% and 40.7% of that in paracancerous hepatic tissue and hepatic tissue in normal mice, respectively.Conclusions: Chol-let-7a, administered either continuously or intermittently, showed effective antitumor efficacy.Chol-let-7a had some off-target effects, such as mild acute hepatitis-like inflammation and non-specific drug-induced kidney injury. The intermittent dosing regimen resulted in less damage than the continuous regimen, while maintaining relatively satisfactory antitumor efficacy, which could be useful for the investigation and possible clinical use of miRNA treatment regimens in the future.