Myocardial gene transfer and long-term expression following intracoronary delivery of adeno-associated virus

Adeno-associated viral vectors (AAV) can direct long-term gene expression in post-mitotic cells. Previous studies have established that long-term cardiac gene transfer results from intramuscular injection into the heart. Cardiac gene transfer after direct intracoronary delivery of AAV in vivo, however, has been minimal in degree, and indirect intracoronary delivery, an approach used in an increasing number of studies, appears to be receiving more attention. To determine the utility of indirect intracoronary gene transfer of AAV, we used aortic and pulmonary artery cross clamping followed by proximal aortic injection of AAV encoding enhanced green fluorescent protein (AAV.EGFP) at 10(11) DNase resistant particles (drp; high-performance liquid chromatography (HPLC)-purified) per rat. Gene expression was quantified by fluorescent microscopy at four time points up to 1 year after vector delivery, revealing 20-32% transmural gene expression in the left ventricle at each time point. Histological analysis revealed little or no inflammatory response and levels of transgene expression were low in liver and undetectable in lung. In subsequent studies in pigs, direct intracoronary delivery into the left circumflex coronary artery of AAV.EGFP (2.64-5.28 x 10(13) drp; HPLC-purified) resulted in gene expression in 3 of 4 pigs 8 weeks following injection with no inflammatory response in the heart. PCR analysis confirmed AAV vector presence in the left circumflex perfusion bed. These data indicate that intracoronary delivery of AAV vector is associated with transgene expression in the heart, providing a means to obtain long-term expression of therapeutic genes.     

CFTR基因在小鼠肠道组织中的差异表达

目的研究肠道组织CFTR基因表达与分泌性腹泻发生的关系。方法选取KM小鼠24只,雌雄各半,随机分为3组（每组8只）：对照组经小鼠腹腔注射0.2 mL生理盐水,实验组小鼠经腹腔注射LPS[6 mg/（kg·bw）]分别作用1 h、8 h,于注射后通过小鼠精神状态、肠道组织形态学判定分泌性腹泻模型的建立,利用荧光定量PCR法检测各段肠道组织CFTR基因的表达。结果 LPS成功诱导小鼠发生了分泌性腹泻;CFTR基因在小鼠十二指肠、空肠、回肠和结肠组织中均有不同的表达丰度,以结肠最高,但各段肠道间差异不显著;与对照组相比,LPS上调了十二指肠、空肠和回肠CFTR基因的转录,下调了结肠CFTR基因的转录。结论提示肠道组织CFTR基因转录水平的上调与LPS诱导分泌性腹泻的发生密切相关,且在各肠段发挥的作用不同,其中空肠在氯离子（Cl-）分泌中发挥主要作用,结肠的作用最弱。     

Modulation of the immune system by Listeria monocytogenes-mediated gene transfer into mammalian cells

In this study, we established a method for Listeria monocytogenes(Lm)-mediated gene transfer into mammalian cells to manipulate the immune response of the host during infection by pathogens. We used the Lm-mediated gene transfer method in an in vivo study to manipulate host immune responses against Leishmania major(L. major )-infection. The injection of Lm modulated the susceptible host into a resistant state against L. major-infection. A more efficient protective effect was obtained with the injection of IL-12-cDNA containing Lm, and the protective effect was stronger than that of the resistant strain. The protective mechanism of Lm-injection against L. major-infection observed here appeared to be a result of the activation of the local immune system by the Lm-mediated gene transfer method. The present study is the first demonstration that a gene introduced into a host by Lm works to modulate the murine host immune response against infections in vivo. Since this system strongly induces Th1 responses and suppresses Th2 responses in infected hosts, the system can be used for controlling infectious diseases and for protection against allergic responses in the future.     

Cystic fibrosis: A look into the future of prenatal screening and therapy

Despite recent guidelines suggesting prenatal screening for carriers of cystic fibrosis (CF) mutations, many physicians do not offer patients this service or even counseling. Some argue that the risks of miscarriage associated with prenatal diagnostic techniques outweigh the benefit of added insight, but with the advent of newer, noninvasive techniques, risks of miscarriage may be significantly lowered. Prenatal diagnosis provides parents the time to prepare for raising a child with CF, and soon, could provide treatment options in utero that could improve quality of life. Here, we describe two of the most promising gene therapy approaches: lentivirus and adenoassociated virus (AAV)‐mediated gene transduction. Thus, prenatal detection and treatment is in a most crucial stage for care of patients with CF. Birth Defects Research (Part C) 105:73–80, 2015. © 2015 Wiley Periodicals, Inc.     

Evaluation of a porcine model for pulmonary gene transfer using a novel synthetic vector

Background

The pig lung, given its gross anatomical, histological and physiological similarities to the human lung, may be useful as a large animal model, in addition to rodents, in which to assess the potential of vectors for pulmonary airway gene transfer. The aim of this study was to assess the utility of the pig lung as a model of gene transfer to the human lung with a synthetic vector system.

Methods

The LID vector system consists of a complex of lipofectin (L), integrin‐binding peptide (I) and plasmid DNA (D). LID complexes containing a β‐galactosidase reporter gene under a CMV promoter or a control plasmid at1 mg/3 ml PBS, or 3 ml buffer, was administered to the right lower lobe ofthe pig lung through a bronchoscope. Pigs were culled at 48 h and lung sections prepared for immunohistochemical and histological analysis. Bronchoalveolar lavage fluid was collected and analysed for TNF‐α by ELISA.

Results

Immunohistochemical staining for the β‐galactosidase reporter gene indicated high efficiency of gene transfer by the LID vector to pig bronchial epithelium with 46% of large bronchi staining positively. There was no evidence for vector‐specific inflammation assessed by leukocytosis and cytokine production.

Conclusions

This study demonstrates the use of the pig for studies of gene transfer in the lung and confirms in a second species the potential of the LID vector for gene therapy of pulmonary diseases such as cystic fibrosis. Copyright © 2002 John Wiley & Sons, Ltd.

     

Effect of polyethylenimine on recombinant adeno-associated virus mediated insulin gene therapy

BACKGROUND: Recombinant adeno-associated virus (rAAV) is becoming a promising vector for gene therapy for type I diabetes. The objective of this study was to investigate the effect of incorporation of polyethylenimine (PEI) on rAAV-mediated insulin gene therapy in vitro and in vivo. METHODS: Recombinant AAV vector, harboring the furin-mutated human insulin and enhanced green fluorescent protein (EGFP) genes, was constructed. The effect of complexation with PEI on rAAV-mediated gene transfer was examined in Huh7 human hepatoma cells. The transgene expression was also examined in streptozotocin (STZ)-induced diabetic C57BL/6J mice by direct administration of rAAV into the livers of the animals, followed by monitoring changes in body weight and blood glucose levels. Secretion of human insulin was determined by radioimmunoassay (RIA) and immunohistochemical staining in the livers. RESULTS: Complexation with PEI was shown to enhance rAAV-mediated transgene expression in Huh7 cells, resulting in higher transduction efficiency and enhanced production of immunoreactive human insulin. Heparin competition assay demonstrated that endocytosis of rAAV-PEI was partially inhibited by heparin. The enhancement of rAAV-mediated transgene expression was also demonstrated in the animals, showing lowering of blood glucose and longer duration of normoglycemia. Immunofluorescent staining of the liver sections demonstrated that PEI increased the uptake of rAAV and enhanced insulin secretion. The enhancement of PEI on rAAV-mediated insulin gene therapy was further confirmed by glucose challenge and a 10-h fasting blood glucose test. CONCLUSIONS: Results obtained in this study demonstrated that incorporation of PEI augmented rAAV-mediated insulin gene transfer and enhanced amelioration of hyperglycemia in the STZ-induced diabetic animals.     

Controlled release of adeno-associated virus from alginate hydrogel microbeads with enhanced sensitivity to ultrasound

Adeno-associated virus (AAV)-based gene therapy holds promise as a fundamental treatment for genetic disorders. For clinical applications, it is necessary to control AAV release timing to avoid an immune response to AAV. Here we propose an ultrasound (US)-triggered on-demand AAV release system using alginate hydrogel microbeads (AHMs) with a release enhancer. By using a centrifuge-based microdroplet shooting device, the AHMs encapsulating AAV with tungsten microparticles (W-MPs) are fabricated. Since W-MPs work as release enhancers, the AHMs have high sensitivity to the US with localized variation in acoustic impedance for improving the release of AAV. Furthermore, AHMs were coated with poly-l -lysine (PLL) to adjust the release of AAV. By applying US to the AAV encapsulating AHMs with W-MPs, the AAV was released on demand, and gene transfection to cells by AAV was confirmed without loss of AAV activity. This proposed US-triggered AAV release system expands methodological possibilities in gene therapy.     

Increased frequency of CFTR gene mutations identified in Indian infertile men with non-CBAVD obstructive azoospermia and spermatogenic failure

High incidence of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is associated with congenital bilateral absence of the vas deferens (CBAVD) and is considered as the genital form of cystic fibrosis (CF). The CFTR gene may also be involved in the etiology of male infertility in cases other than CBAVD. The present study was conducted to identify the spectrum and frequency of CFTR gene mutations in infertile Indian males with non-CBAVD obstructive azoospermia (n = 60) and spermatogenic failure (n = 150). Conspicuously higher frequency of heterozygote F508del mutation was detected in infertile males with non-CBAVD obstructive azoospermia (11.6%) and spermatogenic failure (7.3%). Homozygous IVS(8)-5T allele frequency was also significantly higher in both groups in comparison to those in normal healthy individuals. Two mutations in exon 25 viz., R1358I and K1351R were identified as novel mutations in patients with non-CBAVD obstructive azoospermia. Mutation R1358I was predicted as probably damaging CFTR mutation. This is the first report from the Indian population, emphasizing increased frequency of CFTR gene mutations in male infertility other than CBAVD. Thus, it is suggested that screening of CFTR gene mutations may be required in infertile Indian males with other forms of infertility apart from CBAVD and willing for assisted reproduction technology.     

Combinatorial engineering of a gene therapy vector: directed evolution of adeno-associated virus

BACKGROUND: Viruses are being exploited as vectors to deliver therapeutic genetic information into target cells. The success of this approach will depend on the ability to overcome current limitations, especially in terms of safety and efficiency, through molecular engineering of the viral particles. METHODS: Here we show that in vitro directed evolution can be successfully performed to randomize the viral capsid by error prone PCR and to obtain mutants with improved phenotype. RESULTS: To demonstrate the potential of this technology we selected several adeno-associated virus (AAV) capsid variants that are less efficiently neutralized by human antibodies. These mutations can be used to generate novel vectors for the treatment of patients with pre-existing immunity to AAV. CONCLUSIONS: Our results demonstrate that combinatorial engineering overcomes the limitations of rational design approaches posed by incomplete understanding of the infectious process and at the same time offers a powerful tool to dissect basic viral biology by reverse genetics.     

Lentivirus vector-mediated gene transfer to the developing bronchiolar airway epithelium in the fetal lamb

BACKGROUND: Development of effective and durable gene therapy for treatment of the respiratory manifestations of cystic fibrosis remains a formidable challenge. Obstacles include difficulty in achieving efficient gene transfer to mature airway epithelium and the need to stably transduce self-renewing epithelial progenitor cells in order to avoid loss of transgene expression through epithelial turnover. Targeting the developing airway epithelium during fetal life offers the prospect of circumventing these challenges. METHODS: In the current study we investigated vesicular stomatitis virus glycoprotein (VSVg)-pseudotyped HIV-1-derived lentivirus vector-mediated gene transfer to the airway epithelium of mid-gestation fetal lambs, both in vitro and in vivo. In the in vitro studies epithelial sheet explants and lung organ culture were used to examine transduction of the proximal and more distal airway epithelium, respectively. For the in vivo studies, vector was delivered directly into the proximal airway. RESULTS: We found that even during the early pseudoglandular and canalicular phases of lung development, occurring through mid-gestation, the proximal bronchial airway epithelium was relatively mature and highly resistant to lentivirus-mediated transduction. In contrast, the more distal bronchiolar airway epithelium was relatively permissive for transduction although the absolute levels achieved remained low. CONCLUSION: This result is promising as the bronchiolar airway epithelium is a major site of pathology in the cystic fibrosis airway, and much higher levels of transduction are likely to be achieved by developing strategies that increase the amount of vector reaching the more distal airway after intratracheal delivery.     

Inhibition of corneal neovascularization with endostatin delivered by adeno-associated viral (AAV) vector in a mouse corneal injury model

The use of a recombinant adeno-associated viral (rAAV) vector carrying endostatin gene as an anti-angiogenesis strategy to treat corneal neovascularization in a mouse model was evaluated. Subconjunctival injection of recombinant endostatin-AAV was used to examine the inhibition of corneal neovascularization induced by silver nitrate cauterization in mice. The results showed that gene expression in corneal tissue was observed as early as 4 days after gene transfer and stably lasted for over 8 months with minimal immune reaction. Subconjunctival injection of a high-titer rAAV-endostatin successfully inhibited neovascularization. Immunohistchemistry staining of CD 31 and endostatin showed that the treatment significantly inhibits angiogenesis in cornea. We concluded that the rAAV was capable of directly delivering genes to the ocular surface epithelium by way of subconjunctival injection and was able to deliver sustained, high levels of gene expression in vivo to inhibit angiogenesis.     

Engineered recombinant ovomucoid third domain can modulate allergenic response in Balb/c mice model

IgE-mediated allergic reactions to egg white are a serious health problem and ovomucoid being the dominant egg white allergen has been on focus in the past decade. Engineered hypoallergens with reduced reactivity for IgE antibodies are being examined to modulate the allergic response and develop prophylactic allergen vaccines. In this study, we evaluated the immunomodulatory effect of a genetic variant of the third domain of ovomucoid (GMFA) which showed reduced IgE binding with egg allergic patient's sera in comparison to the native form of the third domain of ovomucoid (DIII) in a murine model system. Balb/c mice were injected intraperitoneally with DIII and GMFA antigens. Allergen-specific serum IgG, IgG1, IgG2a, and IgE responses were evaluated using enzyme-linked immunosorbent assay. Splenocyte cytokine levels in the medium of the cultured cells were examined by ELISA and levels of IL-4, INF-gamma, and IL-12 (p70) cytokines were quantified. Neutralization with anti-IL-12 monoclonal antibody was assayed and cytokine levels with respect to GMFA mutant antigen stimulation were measured. GMFA mutant form was found to have significantly reduced levels of specific IgE when compared to the DIII suggesting a mutation-induced abrogation of the IgE binding epitope in mice. The increase in IgG2a levels in GMFA together with the decline of IgE and IgG1 points to a shift from a Th2 response to a Th1 dominated response. The cytokine profile showed a modulation of anti-allergic Th1 phenotype in GMFA from a proallergic Th2 response observed with DIII. Low levels of IL-4 and increased levels of INF-gamma and IL-12 were observed and anti-IL-12 monoclonal antibody restored the levels of IL-4 and suppressed levels of INF-gamma and IL-12 in the GMFA sensitized group. These results indicate that GMFA has a marked suppressive effect on the allergic response of ovomucoid and caused a shift towards a Th1 pathway, thereby modulating the Th1/Th2 cytokine balance and could be used as a potential hypoallergenic candidate for allergen-immunotherapy in the treatment of egg white allergy.     

Successful targeting of the mouse cystic fibrosis transmembrane conductance regulator gene in embryonal stem cells

We wish to construct a mouse model for the human inherited disease cystic fibrosis. We describe here the successful targeting in embryonal stem cells of the murine homologue (Cftr) of the cystic fibrosis transmembrane conductance regulator gene, as the first critical step towards this end. The targeting event precisely disrupts exon 10, the site of the major mutation in patients with cystic fibrosis. The targeted cells are pluripotent and competent to form chimaeras.     

Skeletal muscle-specific expression of human blood coagulation factor Ⅸ rescues factor Ⅸ deficiency mouse by AAV-mediated gene transfer

The efficacy of recombinant adeno-associated virus (AAV) vector to deliver and express human blood clotting factor DC (hFIX) gene in skeletal muscle of coagulation factor IX deficiency mouse strain (FactorIX-knockout) is e-valuated. The muscle creatine kinase enhancer (MCK) and βactin promoter ((3A) were used to drive the hFIX minigene (hFIXml), which was flanked by AAV inverted terminal repeats (ITRs). Following intramuscular injection of high liter (2.5 x 1011 vector genomes/mL) of AAV, increased hFIX expression (256 ng/mL of plasma) was achieved. The time course of hFIX expression demonstrated that the expression level gradually increased over a period of two weeks before anti-hFIX antibodies developed in mouse circulating plasma. Those results provided a promising evidence that rAAV-me-diated gene transfer and skeletal muscle-specific expression of hFIX is a feasible strategy for treating patients for hemophilia B.     

Inflammation-inducible anti-TNF gene expression mediated by intra-articular injection of serotype 5 adeno-associated virus reduces arthritis

BACKGROUND: The tumor necrosis factor (TNF)-alpha plays a central role in rheumatoid arthritis (RA) and current biotherapies targeting TNF-alpha have a major impact on RA treatment. The long-term safety concerns associated with the repetitive TNF blockade prompt optimization of therapeutic anti-TNF approaches. Since we recently demonstrated that intra-articular gene transfer using a recombinant adeno-associated virus serotype 5 (rAAV5) efficiently transduces arthritic joints, we evaluate its effect on collagen-induced arthritis (CIA) when encoding TNF antagonists. METHODS: Recombinant AAV5 vectors encoding the human TNFRp55 extracellular domain fused to the Fc region of mice IgG1 (TR1) or a small molecular weight dimeric human TNFRp75 extracellular domain (TR2), under two different promoters, the CMV or a chimeric NF-kappaB-based promoter inducible by inflammation, were injected into mouse CIA joints. RESULTS: Best protection against arthritis was obtained with the rAAV5 encoding the TR1, as reflected by delayed disease onset, decreased incidence and severity of joint damage. This effect was associated with a transient expression of the anti-TNF agent when expressed under a NF-kappaB-responsive promoter, only detectable during disease flare, while the antagonist expression was rapidly increased and stable when expressed from a CMV promoter. Importantly, using the intra-articular administration of the rAAV5-NF-kappaB-TR1 vector, we observed a striking correlation between local TR1 expression and inflammation. CONCLUSIONS: These findings strongly support the feasibility of improving the safety of anti-TNF approaches for the treatment of arthritis by local rAAV5-mediated gene expression under an inflammation-responsive promoter, able to provide a limited, transient and therapeutically relevant expression of anti-TNF compounds.