Effectiveness of enterovirus A71 vaccine against pediatric HFMD and disease profile of post-vaccination infection.

BACKGROUND: Vaccination has been proven effective against infection with enterovirus A71 (EV-A71) in clinical trials, but vaccine effectiveness in real-world situations remains incompletely understood. Furthermore, it is not clear whether previous vaccination will result in symptom attenuation among post-vaccinated cases. METHODS: Based on long-term data extracted from the only designed referral hospital for infectious diseases, we used a test-negative case-control design and multivariate logistic regression models to analyze the effectiveness of EV-A71 vaccine against hand, foot and mouth disease (HFMD). And then, generalized linear regression models were used to evaluate the associations between prior vaccination and disease profiles. RESULTS: We selected 4883 inpatients for vaccine efficacy estimations and 2188 inpatients for disease profile comparisons. Vaccine effectiveness against EV-A71-induced HFMD for complete vaccination was 63.4 % and 51.7 % for partial vaccination. The vaccine effectiveness was higher among cases received the first dose within 12 months. No protection was observed against coxsackievirus (CV) A6-, CV-A10- or CV-A16-associated HFMD among children regardless of vaccination status. Completely vaccinated cases had shorter hospital stay and disease course compared to unvaccinated cases (P < 0.05). CONCLUSIONS: These findings reiterate the need to continue the development of a multivalent vaccine or combined vaccines, and have implications for introducing optimized vaccination strategies.

Interrelation of Chronic Lung Disease and Cardiovascular Disease Based on Two National Prospective Cohort Studies.

BACKGROUND: Chronic lung diseases (CLDs) and cardiovascular diseases (CVDs) are the main chronic diseases responsible for a considerable burden of disease. This study aimed to estimate the interrelation of CLDs and CVDs using two Chinese national longitudinal cohort studies. METHODS: The China Health and Retirement Longitudinal Study (CHARLS) and Chinese Longitudinal Healthy Longevity Survey (CLHLS) were used in this study with 15,052 and 9,765 participants, respectively. The Cox proportional risk model was used to estimate the interrelation between CLDs and CVDs. Mediating effects were performed to detect possible influencing pathways between CLDs and CVDs. RESULTS: The association of CLDs with CVDs was identified in 1,647 participants (10.9%) with newly diagnosed CVDs in CHARLS and 332 participants (11.6%) in CLHLS. The Cox proportional risk model showed that CLDs were a significant predictor of CVDs (HR 1.49, 95% CI 1.27-1.76) after adjusting for covariates, and the hazard ratios of stroke and CVDs excluding stroke were (HR 1.02, 95% CI 0.79-1.31) and (HR 1.76, 95% CI 1.46-2.13), respectively. These association were mediated by body mass index (BMI) and Center for Epidemiological Studies Depression Scale (CES-D-10) scores. No significant association was found in CHARLS and CLHLS regarding CVDs with CLDs. In CHARLS, CVDs was a significant predictor of CLDs (HR 1.40, 95% CI 1.09-1.79). CONCLUSIONS: Chronic lung disease was associated with increased incidence of CVDs in middle-aged and older people in the community population and vice versa. Body mass index and depressive symptoms might be mediated by the effect of CLD on CVD.

Bright, small sizes and hydro-dispersive NIR persistent luminescence nanoparticles modified with Si and amino groups for enhanced bioimaging.

Near-infrared (NIR) persistent luminescence nanoparticles (PLNPs) with high brightness, small sizes, good hydro-dispersivity, and intrinsic surface-functional groups are desirable in biological applications. In this work, Cr3+-doped zinc gallogermanates Zn1+xGa2-2xGexO4:Cr (ZGGC) PLNPs were hydrothermally synthesized via 3-aminopropyltriethoxysilane (APTES) as an additive, or APTES and cetyltrimethylammonium bromide (CTAB) as two co-additives. Addition of APTES not only dramatically enhances the 696 nm NIR luminescence intensity, but also obviously decreases the particle size and introduces amino groups. In particular, thex= 0.1 series ZGGC (ZGGC0.1) with the addition of n moles equivalent APTES (ZGGC0.1-nA) had smaller particle sizes than thex= 0.2 counterpart (ZGGC0.2-nA). The NIR afterglow intensities increased with the APTES introduction. The ZGGC0.2-2.5A sample (also named as ZGGC, Si, -NH2) exhibited maximum luminescence intensities both in solid and aqueous states. With APTES, Si atom is doped and -NH2groups are modified, the trap depth and density become larger, and the afterglow intensities and decay time are significantly enhanced. More notably, co-addition of CTAB (ZGGC0.2-2.5A-C) (also named as ZGGC, Si, -NH2') further enhances hydro-dispersivity and luminescence intensity, decreases particle sizes, and results in more prominent amino groups. The trap density is drastically higher than that without CTAB (i.e. ZGGC0.2-2.5A). Change of Cr3+microenvironment in the crystal and more defects introduction contribute to the enhanced brightness. As expected, the ZGGC,Si,-NH2' PLNPs possess excellent biocompatibility, deep tissue penetration and distinguished bioimaging properties, and rechargeability with orange LED light. The ZGGC,Si,-NH2' PLNPs should provide to be an excellent nanomaterial for various functionalization and bioimaging applications.

Enhancing the antitumor immunosurveillance of PD-L1-targeted gene therapy for metastatic melanoma using cationized Panax Notoginseng polysaccharide.

Improved curative effects with reduced toxicity has always been the ultimate goal of gene delivery vectors for tumor immunotherapy. Panax notoginseng polysaccharide (PNP), a natural plant-derived macromolecule, not only has antitumor immune activity but also has the typical structural characteristics useful for gene delivery. In this work, positively charged polyethyleneimine (PEI) was directly grafted to the backbone of PNP to induced its charge reversal and generate a functional gene vector (PNP-PEI). Moreover, a short hairpin RNA targeting the programmed death-ligand 1 (PD-L1) was loaded into PNP-PEI to generate a potentially therapeutic nanoparticle (PNP-PEI/shPD-L1). In vitro and in vivo experiments demonstrated that PNP-PEI could efficiently carry the therapeutic shPD-L1 into tumor cells and that PNP-PEI/shPD-L1 could significantly inhibit the expression of PD-L1 and growth of B16-F10 cells. Noteworthily, treatment with PNP-PEI reversed the phenotype of macrophages from M2 to M1 subtype and promoted dendritic cell maturation, which encouraged the host immunity and enhanced the therapeutic antitumor effects. In summary, this study describes a PNP-based gene delivery vector and highlights the beneficial immunopotentiating therapeutic outcomes of PNP-PEI for tumor immunotherapy.

Molecular epidemiology and clinical features of hand, foot and mouth disease requiring hospitalization after the use of enterovirus A71 inactivated vaccine in chengdu, China, 2017-2022: a descriptive study.

Three inactivated enterovirus A71 (EV-A71) vaccines have been widely vaccinated among children in the targeted age group in mainland China since mid-2016. However, comprehensive virological surveillance of hand, foot and mouth disease (HFMD) over multiple years after the use of EV-A71 vaccines has rarely been conducted. Using long-term data extracted from the Public Health and Clinical Center of Chengdu, we described the clinical, aetiological, and epidemiological characteristics of HFMD inpatients after the use of EV-A71 vaccines from 2017 through 2022. A total of 5115 patients were selected for analysis with a male-to-female ratio of 1.63:1 and were mostly under 5 years of age (97.6%). Among these cases, 4.3% presented with severe symptoms, and 4.1% of severe cases experienced significant complications. EV-A71 was no longer the major serotype for laboratory-confirmed HFMD, responsible for 15.6% of severe cases and 1.2% of mild cases. A significant downwards trend of EV-A71 infections was observed after the use of EV-A71 vaccines (P for trend < 0.001). Coxsackievirus A6 was the predominant pathogen, accounting for 63.5% of mild cases and 36.2% of severe cases. Coxsackievirus A10 (CV-A10) and A16 were sporadically detected, and an upwards trend was observed in the proportion of CV-A10 infections. This study provides baseline molecular epidemiology for the evaluation of EV-A71 vaccination impact and potential serotype replacement based on HFMD inpatients. Additional nationwide and population-based epidemiologic and serologic studies are essential to elucidate HFMD dynamics after the use of EV-A71 vaccines, and to inform public health authorities to introduce optimized intervention strategies.

Relationship between meteorological factors, air pollutants and hand, foot and mouth disease from 2014 to 2020.

BACKGROUND: Meteorological factors and air pollutants have been reported to be associated with hand, foot, and mouth disease (HFMD) epidemics before the introduction of vaccine. However, there is limited evidence for studies with long-term dimensions. METHODS: We collected the daily HFMD counts, weather and air pollution data from 2014 to 2020 in Chengdu. Distributed lag non-linear models (DLNM) were used to assess the associations of meteorological factors and air pollutants on HFMD cases. RESULTS: From 2014-2020, high relative humidity and precipitation and extremely high and low levels of PM10, O3, SO2 and CO increased the risk of HFMD. In pre-vaccination period, extreme high and low temperatures, PM10 and NO2, low precipitation and high concentrations of PM2.5 and O3 significantly increase the risk of HFMD; In post-vaccination period, high relative humidity and low level of CO can significantly increase the incidence of HFMD; During the period of COVID-19, only low temperature will significantly increase the risk of HFMD; Low concentration of air pollutants has the greatest impact on the 6-14 age group, while the high concentration of air pollutants has the greatest impact on the 0-1 age group. CONCLUSIONS: Our study suggest that high relative humidity and precipitation and extremely high and low levels of PM10, O3, SO2 and CO increased the risk of HFMD from 2014 to 2020. The results of this study provide a reference for local authorities to formulate intervention measures and establish an environment-based disease early warning system.

Dynamic changes of viral load and the duration of viral shedding in patients with hand, foot and mouth disease: a protocol for longitudinal study.

BACKGROUND: The duration of virus shedding is necessary for determining the infectious period. But there were few quantitative studies on the changes of viral load and the law of the viral shedding in hand foot and mouth disease (HFMD) patients has not yet been clarified. METHODS: This study will prospectively recruit coxsackievirus A10 (CV-A10), coxsackievirus A16 (CV-A16) and coxsackievirus A6 (CV-A6) infected inpatients from January 2022 to December 2022. A series of samples and questionnaire information will be collected regularly to establish the dynamic function relationship between time and viral load changes and a Bayesian multilevel model will be constructed to clarify the evolvement rules which reflect the dynamic changes of viral load and the duration of viral shedding in patients with HFMD. DISCUSSION: The results of this study is expected to further clarify the evolvement rules which reflect the dynamic changes of viral load and the duration of viral shedding in HFMD patients under the influence of related factors. It can also provide important evidence for the scientific definition of the infectious period and isolation period of HFMD in China.

Spatiotemporally co-delivery of triple therapeutic drugs via HA-coating nanosystems for enhanced immunotherapy.

There is growing empirical evidence that certain types of chemotherapy and phototherapy trigger immunogenic cell death and enhance the therapeutic anticancer efficacy of genetic immunotherapy. However, the main challenge is spatiotemporally co-delivering different drugs to maximize the therapeutic index of the combination therapy. In this study, a drug delivery system (HTCP-Au/shPD-L1/DOX) was designed with a polysaccharide-wrapped shell and a condensed DNA core. To construct the HTCP-Au vector, dodecyl side chains with a polyethylenimine (PEI) head were grafted onto hyaluronic acid, and AuNPs were grafted via Au-S bonds. During drug loading, PEI arrested shRNA plasmid DNA targeting programmed cell death ligand 1 (shPD-L1) via electrostatic interactions. It also formed a PEI-DNA core that was automatically enclosed when aliphatic hydrocarbons pulled the hyaluronic acid backbone. A hydrophobic interlayer consisting of dodecyl bridge chains between the PEI-DNA core and the hyaluronic acid shell was required to accommodate hydrophobic doxorubicin. In vitro and in vivo assays demonstrated that this core-shell drug delivery system could efficiently load and transport three different drugs and effectively target tumors. Moreover, it could activate the immune system, thereby providing promising therapeutic efficacy against tumor growth and metastasis.

The Role of Blood Pressure in Carotid Plaque Incidence: Interactions With Body Mass Index, Age, and Sex-Based on a 7-Years Cohort Study.

Background: Although high blood pressure (BP) is a risk factor for carotid plaque, its long-term prognostic value might be underestimated due to its confounding interactions with BMI, age, and gender. Therefore, we conducted a 7-year prospective cohort study to evaluate the prognostic value of BP for the incidence of carotid plaque. Methods: The subjects enrolled in 2011 were free of carotid plaque at baseline and were followed up in 2018. Multivariate Cox proportional-hazards models were used to evaluate the association between BP and carotid plaque incidence. Results: During the follow-up study, the incidence of carotid plaque was 36.5%. The significant positive linear trend showed that subjects with higher BP levels at baseline were more likely to develop carotid plaques at the end. Especially in the female subpopulation, after confounders being adjusted, the carotid plaque was associated with higher BP (adjusted HR 1.52, 95% CI 1.02-2.26), pulse pressure (PP) (adjusted HR 1.15, 95% CI 0.76-1.75), and mean arterial pressure (MAP) (adjusted HR 1.44, 95% CI 1.00-2.08). The adjusted HRs of hypertension, PP, and MAP (HR 27.71, 95% CI 2.27-338.64; HR 14.47, 95% CI 1.53-137.18; HR 9.97, 95% CI 1.29-77.28) were significantly higher after the potential antagonistic interactions between BP categorical indicators and age being adjusted, respectively. Conclusion: High BP indicators might be associated with higher HRs of carotid plaque after adjusting interactions between BP indicators and BMI, age, and gender, which suggests that the incidence of carotid plaque in female adults with high BP indicators might increase significantly with the increase of age.

Chronic Lung Diseases and the Risk of Depressive Symptoms Based on the China Health and Retirement Longitudinal Study: A Prospective Cohort Study.

Chronic lung diseases (CLDs) can reduce patients' quality of life. However, evidence for the relationship between CLD and occurrence with depressive symptoms remains unclear. This study aims to determine the associations between CLD and depressive symptoms incidence, using the data from the China Health and Retirement Longitudinal Study (CHARLS). CLD was identified via survey questionnaire and hospitalization. The follow-up survey was conducted in 2018 and depressive symptoms were assessed by the 10-item Center for Epidemiological Studies Depression Scale (CES-D-10). A total of 10,508 participants were studied with an average follow-up period of 3 years. A total of 2706 patients (25.8%) with newly diagnosed depressive symptoms were identified. The standardized incidence rate of depressive symptoms in baseline population with and without chronic pulmonary disease was 11.9/100 and 8.3/100 person-years, respectively. The Cox proportional risk model showed that CLD was a significant predictor of depressive symptoms (HR: 1.449, 95% CI: 1.235-1.700) after adjusting for covariates, and the HRs of depressive symptoms were higher in those participants with current smoking (HR: 1.761, 95% CI: 1.319-2.352), men (HR: 1.529, 95% CI: 1.236-1.892), living in rural areas (HR: 1.671, 95% CI: 1.229-2.272), with dyslipidemia (HR: 1.896, 95% CI: 1.180-3.045), and suffering from comorbidity (HR: 1.518, 95% CI: 1.104-2.087) at baseline survey. CLD was an independent risk factor of depressive symptoms in China. The mental health of CLD patients deserves more attention.

The Role of BMI and Blood Pressure in the Relationship Between Total Cholesterol and Disability in Chinese Centenarians: A Cross-Sectional Study.

Background: Lower serum lipid metabolism might be associated with the decline of activity of daily living in the extreme longevity group. However, studies on models and possible paths of this correlation between total cholesterol (TC) and disability in centenarians are scarce. The aim of this study was to verify this correlation and explore the mediating effect of BMI and blood pressure on this relationship in Hainan centenarians. Methods: We conducted a cross-sectional analysis of 1002 centenarians from the China Hainan Centenarians Cohort Study (CHCCS). Data on demographics, anthropometry data, lifestyle, and TC levels were collected through interviews, physical examinations, and laboratory tests. The Barthel index and Lawton index, measuring the disability status, were used to estimate the activity of daily living (ADL) and instrumental activity of daily living (IADL). A multivariable logistic regression model was used to explore the correlation between disability and TC levels. Mediation analyses were used to explore the both direct and indirect effects of TC level on disability. Results: After adjusting for covariates, with 1 mmol/L increment in TC, the adjusted odds ratios (ORs) of ADL severe disability and ADL moderate & severe disability were 0.789(95%CI: 0.650-0.959) and 0.822(95%CI: 0. 0.699-0.966), respectively. There was a significant declining trend in the prevalence of different types of disability with increment in TC. The correlation was more pronounced among Hainan female centenarians. In the analysis of mediating effect among the female population, BMI significantly mediated the effect of TC levels on different types of disability. BMI and SBP, as chain mediators, multiply and chain mediated the effect of TC levels on IADL. Conclusion: Low TC levels might be correlated with a higher frequency of disability in female centenarians, and this correlation might be mediated by BMI and blood pressure.

Endoplasmic reticulum targeted fluorescent probe for the detection of hydrogen sulfide based on a twist-blockage strategy.

Hydrogen sulfide (H2S) is very important for humans and is involved in many physiological processes. Here, we designed and reported a new naked-eye colorimetric fluorescent probe Z1 for detecting H2S in absolute HEPES solution. The fluorescence intensity, after the reaction of the probe and H2S, is about 32 times that of the probe alone. When the concentration of H2S is 0-100 µM, the detection limit (DL) is rather low at about 0.15 µM (3σ/slope). The response mechanism is based on the leaving of the 2,4-dinitrobenzene moiety, followed by intramolecular cyclization to give a fluorescent iminocoumarin-benzothiazole group. Moreover, Z1 was applied to endogenous and exogenous H2S imaging in living cells. The high overlap coefficient proved that probe Z1 has good ER-tracker localization in living cells.

Multiclass analysis of 25 veterinary drugs in milk by ultra-high performance liquid chromatography-tandem mass spectrometry.

The aim of this work was to develop a simple and rapid analytical method for the simultaneous determination of a wide range of drug residues in milk by UHPLC-MS/MS. A total of 25 typical veterinary drugs investigated belong to six families including ß-lactams, quinolones, ß-agonists, phenicols, glucocorticoids and nitrofurans. The samples were extracted by acetonitrile and defatted with n-hexane twice. Electrospray ionization and positive/negative polarity switching were utilized for the analysis of 25 veterinary drugs in a single chromatographic run. The linearity, recovery, precision and matrix effects of the method were fully validated. The intra- and inter-precision were in the range of 1.7-11.1% and 2.5-10.4%, respectively. The average recoveries ranged from 65.9% to 123.5% with RSD less than 10.8% at three concentration levels. The proposed method was demonstrated to be simple, economical and reliable for the fast monitoring of these drug residues in milk samples.

A novel fluorescent long-chain fatty acid-substituted dye: labeling and biodegrading of Microthrix parvicella.

Microthrix parvicella (M. parvicella) is a filamentous bacterium that induces bulking in activated sludge. Here, we used the affinity of long-chain fatty acids (LCFA) for M. parvicella to create a novel fluorescent probe of carbazole modified by LCFA. The structure was characterized by 1H NMR spectroscopy and mass spectrometry. The spectral properties, photostability, and hydrophobic properties of the probe were also characterized. Fluorescent-labeling results showed that it can label M. parvicella in situ and could be biodegraded via metabolism. The stable docking mode of carbazole probes with different fatty acid chains and lipases was also docked by the density functional tight-binding (DFTB) method.

Photoactivatable RNAi for cancer gene therapy triggered by near-infrared-irradiated single-walled carbon nanotubes.

The efficacy of RNA interference (RNAi)-based cancer gene therapy is limited by its unexpected side effects, thus necessitating a strategy to precisely trigger conditional gene knockdown. In this study, we engineered a novel photoactivatable RNAi system, named as polyetherimide-modified single-wall carbon nanotube (PEI-SWNT)/pHSP-shT, that enables optogenetic control of targeted gene suppression in tumor cells. PEI-SWNT/pHSP-shT comprises a stimulus-responsive nanocarrier (PEI-SWNT), and an Hsp70B'-promoter-driven RNAi vector (pHSP-shT). In response to near-infrared (NIR) light irradiation, heating of PEI-SWNT in breast MCF-7 cells triggered gene knockdown targeting human telomerase reverse transcriptase through RNAi, with the gene-knockdown activity capable of being switched off by extinguishing the NIR. Furthermore, we demonstrated that the photoactivatable RNAi system exhibited higher antitumor activity by combining gene therapy and photothermal therapy, both in vitro and in vivo. Optogenetic control of RNAi based on an NIR-activated nanocarrier will potentially facilitate improved understanding of molecular-targeted gene therapy in human malignant tumors.

Lysosomal-Targeted Two-Photon Fluorescent Probe to Sense Hypochlorous Acid in Live Cells.

A two-photon reversible fluorescent probe L1 was designed and synthesized. The fluorescence intensity of the probe solution was strong, while the fluorescence of the solution was obviously quenched and the color of the solution was changed after the addition of hypochlorous acid, indicating this is "naked-eye sensor" for the detection of HClO. The probe showed great selectivity for hypochlorous acid over other reactive oxygen species (ROS) and anions. Fluorescence titration experiments showed that the probe has a low detection limit of 0.674 µM. Because of a morpholine group introduced to the naphathalimide framework, probe L1 was successfully applied to detect intracellular HClO in lysosome.

Nanoparticle siRNA against BMI-1 with a Polyethylenimine-Laminarin Conjugate for Gene Therapy in Human Breast Cancer.

The B-cell-specific Moloney leukemia virus inset site 1 gene (BMI-1) has attracted considerable attention in recent years because of its key role in breast cancer development and metastasis. The downregulation of BMI-1 expression via small interfering RNA (siRNA) effectively inhibits tumor growth. However, the successful application of this therapy is limited by the unavailability of an appropriate vector for siRNA transfer. Therefore, this study aimed to construct a novel laminarin-based nonviral gene transfer vector to carry a constructed BMI-1-targeting siRNA and to investigate the in vitro and in vivo antitumor effects of this siRNA on breast cancer cells. To enhance the siRNA-carrying capacity, we introduced polyethylenimine (PEI) to laminarin's surface via N,N'-carbonyldiimidazole, which produced the cationic PEI-modified laminarin conjugate nLP. Subsequent in vitro experiments indicated that nLP not only formed a nanoparticle with a diameter of 200 nm through electrostatic interactions with siRNA but also showed high efficiency (95.0%) in the delivery siRNA to MCF-7 cells. The nanoparticle targeting BMI-1 (nLP/siBMI-2) reduced BMI-1 expression in breast MCF-7 cells by 90.9% reduction. An in vivo tumor suppression experiment demonstrated that the nLP/siBMI-2 nanoparticle had relatively low toxicity and good gene-therapeutic efficacy, with a tumor inhibition rate of 46.6%.

Liver-specific gene therapy of hepatocellular carcinoma by targeting human telomerase reverse transcriptase with pegylated immuno-lipopolyplexes.

The purpose of this study is to explore the possibility and feasibility of liver-specific gene therapy. A shRNA expression plasmid against human telomerase reverse transcriptase (hTERT) was constructed under the control of liver-specific promoter apolipoprotein A-I (ApoAI), designated as pApoAI-shTERT, and its liver-specific cytotoxicity and inhibition of telomerase activity were first evaluated in different cell lines, and its therapeutic effect was further studied in SMMC-7721 human liver tumor-bearing mice in vivo. The results showed that compared to pU6-shTERT, a shRNA expression plasmid against hTERT under the control of U6 promoter, pApoAI-shTERT only significantly diminished the cell viability in the telomerase positive hepatocarcinoma cells and showed no cytotoxicity in the telomerase negative cell lines as well as in the telomerase positive cell line of non-liver origin. Besides, pApoAI-shTERT only significantly reduced telomerase activity in the telomerase positive cell lines of liver origin. Intravenous administration of pegylated immuno-lipopolyplexes (PILP) formulated green fluorescent protein (GFP) expression plasmid under the control of ApoAI into liver tumor-bearing mice resulted in restricted GFP expression in liver and liver tumor. The treatment of pApoAI-shTERT formulated as PILP caused a 56% increase in the life span of SMMC-7721 tumor-bearing mice in vivo relative to the control, which was in agreement with the reduced tumor size and down-regulated hTERT mRNA level in the tumors. We conclude that the vector pApoAI-shTERT was able to cause liver-specific and hTERT target-specific cytotoxicity, and utilizing PILP to deliver pApoAI-shTERT is a promising strategy for liver-specific gene therapy.

Construction, modification and evaluation of apolipoprotein A-I promoter-driven shRNA expression vectors against hTERT.

Liver-specific gene knockdown cannot be achieved by short hairpin RNA (shRNA) generated by RNA polymerase III promoter. Here we constructed, modified and evaluated apolipoprotein A-I (ApoA-I) promoter-driven shRNA expression vectors against human telomerase reverse transcriptase (hTERT) in SMMC-7721 cells. The roles of the cis-acting hammerhead ribozyme and the specific pausing site MAZ, the liver-specific promoter ApoA-I, as well as SV40 and CMV enhancers were first individually evaluated, and then they were incorporated to construct a liver-specific shRNA expression plasmid against hTERT, and the inhibitory effects on hTERT were examined in SMMC-7721 cells. The results showed that the introduction of the cis-acting hammerhead ribozyme and the specific pausing site MAZ did not change gene knockdown efficiency significantly, but eliminated the off-target effect. Green fluorescent protein (GFP) expressing plasmid under the control of ApoA-I promoter can produce liver-specific GFP expression, but at a much lower level compared to the CMV promoter. The CMV or SV40 enhancer-modified ApoA-I promoter caused a four or two folds increase in mRNA expression of GFP relative to ApoA-I alone, respectively. The liver-specific shRNA expression plasmid against hTERT under the control of CMV enhancer-modified ApoA-I promoter with the sequences of the cis-acting hammerhead ribozyme and MAZ, induced significant inhibitory effect on hTERT at both mRNA and protein levels in SMMC-7721 cells. Therefore, liver-specific gene therapy is made possible by utilizing shRNA expression vector under the control of CMV enhancer-modified ApoA-I promoter.

Liver-specific expression of an exogenous gene controlled by human apolipoprotein A-I promoter.

Liver-specific gene therapy is advantageous to minimize the possible adverse effects caused by non-target gene expression. The CMV promoter of the enhanced green fluorescent protein (EGFP) expressing plasmid pCMV-EGFP was replaced with the liver-specific promoter apolipoprotein A-I (ApoAI) generating pApoAI-EGFP plasmid. In vitro expression experiments performed in various cell lines including HepG2, SMMC-7721, MCF7, ACC-2 and Lo2 indicated that pCMV-EGFP treatment caused gene expression in all the cell lines, whereas pApoAI-EGFP treatment only induced EGFP expression in cells of liver origin including the liver cancer cells HepG2 and SMMC-7721 and the normal liver cells Lo2. Either pCMV-EGFP or pApoAI-EGFP was formulated as pegylated immuno-lipopolyplexes (PILP), a novel and efficient gene delivery system. Following intravenous administration of the PILP in H22 tumor-bearing mice, there was significant EGFP expression in liver, tumor, spleen, brain and lung in the pCMV-EGFP treated mice, whereas in the pApoAI-EGFP treated mice there was only gene expression in liver and tumor and the non-liver organ gene expression was eliminated. This study suggests that the use of the PILP technology and liver-specific promoter enables efficient and liver-specific expression of an exogenous gene.