Effect of intratracheal adenoviral vector administration on lung development in newborn rats

Local overexpression of genes that promote lung defense or repair may be helpful in protecting the immature neonatal lung from injuries, but whether the vectors used to administer these genes affect physiological postnatal lung growth has not been investigated. We explored the effect on alveolarization of E1-deleted Adnull vector (Ad5-LMP-null) given intratracheally to 3-day-old rats. Three Adnull doses were evaluated 10(8), 5 x 10(8), and 10(9) TCID(50). Lung morphometry on day 21 showed significant growth disorders with the two higher doses. With 5 x 10(8) TCID(50), absolute lung volume increased significantly (+16%), as did absolute (+20%) and specific (+32%) alveolar airspace volumes, whereas alveolar surface density decreased by 13% (p < 0.009 for all parameters). Lung inflammation was mild, nonsignificant, and occurred mainly with the highest Adnull dose, indicating that it was unlikely to contribute to our results. Adnull instillation induced a significant#10; decrease in terminal bronchiolar cell proliferation as evaluated by proliferating cell nuclear antigen immunostaining (p = 0.02), as well as a 23% decrease in absolute parenchyma elastic fiber length (p = 0.02). Furthermore, lung tropoelastin mRNA content decreased by 25% (p < 0.02). In conclusion, E1-deleted adenoviral vectors can induce lung growth disorders when instilled into the airways of neonatal rats. Interactions with lung matrix turnover may be the main explanation to these deleterious effects.     

Gender differences in serotype 2 adeno-associated virus biodistribution after administration to rodent salivary glands

Salivary glands (SGs) have proven useful targets for clinical applications of gene therapeutics. In this toxicology and biodistribution study, which conforms to U.S. Food and Drug Administration Good Laboratory Practice regulations, four doses (10(7)-10(10) particles) of a serotype 2 adeno-associated viral (AAV2) vector encoding human erythropoietin were directly administered to the right submandibular gland of male and female BALB/c mice (n = 21 per gender dose group). Control-treated (saline administered; n = 66) and vector-treated (n = 168) animals did not differ in clinical appearance, morbidity and mortality rates, food and water consumption, weight gain ratios, and final weight. Clinical hematology values also were unaffected by AAV2 administration except for parameters influenced by the expression of the recombinant protein (e.g., hematocrit). Mice were killed on days 3, 30, 55, and 92. No major vector-related toxicity was uncovered after complete pathology and histopathology review. However, a significant gender-related difference in vector biodistribution was revealed by quantitative polymerase chain reaction. In male mice vector (group receiving 10(10) particles/animal) effectively transduced, and was primarily confined within, the SGs (i.e., approximately 800 times more copies in SGs than in liver; day 3) and long lived. In contrast, in female mice, SG transduction was less efficient (260-fold less than in males; day 3) and short lived, and vector was disseminated widely via both the bloodstream (SG:liver copy ratio, approximately 1) and saliva (30-fold greater than in males). The observed vector biodistribution is likely due to differences in AAV2 receptor targets and structural differences affecting SG integrity. Sexual dimorphism is a factor of major significance that could potentially affect gene therapy clinical applications in SGs.     

Oral administration of recombinant adeno-associated virus elicits human immunodeficiency virus-specific immune responses

Oral vaccines can induce both systemic and mucosal immunity. Mucosal immunity, especially regional cell-mediated immunity, plays an important role in protecting individuals from infectious diseases such as acquired immunodeficiency syndrome. In this study, a recombinant adeno-associated virus vector expressing human immunodeficiency virus type 1 env gene (AAV-HIV) was orally administered to BALB/c mice. Systemic and regional immunity was induced in the mice. Furthermore, the immunization significantly reduced viral load after an intrarectal challenge with a recombinant vaccinia virus expressing HIV env gene. Moreover, we also show that dendritic cells might contribute to the AAV-HIV vector-induced immune responses.     

Inadvertent Methylergonovine administration to a newborn

Previous case reports describe the inadvertent administration of methylergonovine to newborns resulting in rare, life-threatening events including neonatal death. To our knowledge, no case reports exist detailing inadvertent methylergonovine administration in the emergency medicine literature. A newborn infant presented to the emergency department (ED) at hour five of life following methylergonovine administration with periods of apnea and cyanosis. The infant required intubation, mechanical ventilation, and a seven day neonatal intensive care stay. This rare case describes the potential for this error to occur in the community and heightens the vigilance of emergency medicine providers when caring for newborns in their first hours of life.     

气管内给药对大鼠气管黏膜和肺组织影响的研究

目的 研究气管内给药对大鼠气管黏膜及肺组织可能造成的损伤及修复情况.方法 以大鼠为研究对象,经气管穿刺注射生理盐水、利多卡因和丁胺卡那霉素,通过扫描电镜观察气道上皮的超微结构变化,并观察大鼠细支气管黏膜和肺泡上皮细胞的病理改变.结果 局部使用上述3种药物2h后,气管黏膜纤毛细胞排列紊乱、水肿.24h后生理盐水和利多卡因组大鼠气管黏膜损害程度及损害面积明显减轻,48～72 h后基本恢复正常,而丁胺卡那霉素组则到72 h后才开始逐渐恢复.三组大鼠注射药物48h后细支气管出现上皮细胞水肿、排列紊乱,细支气管及肺泡组织周围炎细胞浸润明显,72 h后逐渐恢复.结论 气管内局部使用生理盐水、利多卡因和丁胺卡那霉素,均可造成大鼠气管黏膜和肺泡组织的急性损伤,以丁胺卡那霉素最为明显,但损伤均具有可逆性.     

Long-term efficacy of adeno-associated virus serotypes 8 and 9 in hemophilia a dogs and mice

We reported total correction of blood coagulation plasma factor VIII (FVIII) activity, using adeno-associated virus serotype 8 (AAV8) vectors for liver-specific gene transfer in hemophilia A mice. We now show, irrespective of immunosuppression or route of administration, total long-term correction of hemophilia A mice with pseudotyped AAV8 and AAV9 vectors. We delivered two FVIII vectors, one expressing canine heavy chain and the other expressing canine light chain. Interestingly, when these vectors were given by hepatic portal vein to hemophilia A dogs, only modest FVIII levels were seen despite the species-specific transgene. No dogs treated developed FVIII inhibitors. However, of three dogs treated with AAV8 vector, the single male, given 1.25 x 10(13) genome copies per vector per kilogram (GC/vector/kg), maintained a level of >4.5% for more than 2 years. In contrast, the two female dogs expressed only 2% FVIII activity despite receiving higher doses of 1.52 x 10(13) and 3 x 10(13) GC/vector/kg, respectively. On the other hand, a male dog treated with AAV9 vector at a low dose (6 x 10(12) GC/vector/kg) maintained FVIII levels of 2-2.5% of normal without bleeding for 200 days (observation ongoing). Although hemophilia A mice were not predictive of vector efficacy in dogs, the two treated male dogs became symptom-free for long periods. Even so, translation of these robust vectors either in appropriate large animals or human beings remains challenging.     

Immune responses to adeno-associated virus and its recombinant vectors

Recombinant adeno-associated virus (rAAV) vectors have emerged as highly promising for use in gene transfer for a variety of reasons, including lack of pathogenicity and wide host range. In addition, all virus-encoded genes have been removed from standard rAAV vectors, resulting in their comparatively low intrinsic immunogenicity. For gene replacement strategies, transgenes encoded by rAAV vectors may induce less robust host immune responses than other vectors in vivo. However, under appropriate conditions, host immune responses can be generated against rAAV-encoded transgenes, raising the potential for their use in vaccine development. In this review, we summarize current understanding of the generation of both undesirable and beneficial host immune responses directed against rAAV and encoded transgenes, and how they might be exploited for optimal use of this promising vector system.     

Production of recombinant adeno-associated virus vectors

Recombinant adeno-associated virus (rAAV) is a prototypical gene therapy vector characterized by excellent safety profiles, wide host range, and the ability to transduce differentiated cells. Numerous rAAV-based vectors providing efficient and sustained expression of transgenes in target tissues have been developed for preclinical studies. Interest in rAAV has been driven by advances in production methods originally developed for rAAV serotype 2 vectors and expanded to include alternative serotypes. The transition to clinical trials is dependent on the development of scalable production methods of Good Manufacturing Practice-grade vectors described in this review.     

Receptor targeting of adeno-associated virus vectors

Adeno-associated virus (AAV) is a promising vector for human somatic gene therapy. However, its broad host range is a disadvantage for in vivo gene therapy, because it does not allow the selective tissue- or organ-restricted transduction required to enhance the safety and efficiency of the gene transfer. Therefore, increasing efforts are being made to target AAV-2-based vectors to specific receptors. The studies summarized in this review show that it is possible to target AAV-2 to a specific cell. So far, the most promising approach is the genetic modification of the viral capsid. However, the currently available AAV-2 targeting vectors need to be improved with regard to the elimination of the wild-type AAV-2 tropism and the improvement of infectious titers. The creation of highly efficient AAV-2 targeting vectors will also require a better understanding of the transmembrane and intracellular processing of this virus.     

Pulmonary retention of free and liposome-encapsulated tobramycin after intratracheal administration in uninfected rats and rats infected with Pseudomonas aeruginosa.

The pulmonary residence time of free and liposome-encapsulated tobramycin was studied with uninfected rats and rats infected with Pseudomonas aeruginosa. Chronic infection in lungs was established by intratracheal administration of 10(8) CFU of P. aeruginosa PA 508 prepared in agar beads. After 3 days, a single dose (300 micrograms) of free or liposome-encapsulated tobramycin was given intratracheally to both infected and uninfected rats. At various time intervals (0.25 to 16 h) after drug instillations, the remaining tobramycin was evaluated in blood, lungs, and kidneys by a microbiological assay. Intratracheal instillation of liposome-encapsulated tobramycin resulted in high and sustained levels of tobramycin in lungs of uninfected and infected rats over the 16-h period studied; however, the tobramycin levels were two times higher in uninfected rats. There was no tobramycin detected in the blood or kidneys from these animals. In contrast, the intratracheally instilled free tobramycin was cleared within 3 and 1 h from the lungs of uninfected and infected animals, respectively. These data suggest that the encapsulation of tobramycin in liposomes can result in a significant increase of its residence time within lungs. This study also shows that pulmonary infection was associated with a lowering of tobramycin levels in lungs.     

Early, sustained efficacy of adeno-associated virus vector-mediated gene therapy in glycogen storage disease type Ia

The deficiency of glucose-6-phosphatase (G6Pase) underlies life-threatening hypoglycemia and growth retardation in glycogen storage disease type Ia (GSD-Ia). An adeno-associated virus (AAV) vector encoding G6Pase was pseudotyped as AAV8 and administered to 2-week-old GSD-Ia mice (n = 9). Median survival was prolonged to 7 months following vector administration, in contrast to untreated GSD-Ia mice that survived for only 2 weeks. Although GSD-Ia mice were initially growth-retarded, treated mice increased fourfold in weight to normal size. Blood glucose was partially corrected by 2 weeks following treatment, whereas blood cholesterol normalized. Glucose-6-phosphatase activity was partially corrected to 25% of the normal level at 7 months of age in treated mice, and blood glucose during fasting remained lower in treated, affected mice than in normal mice. Glycogen storage was partially corrected in the liver by 2 weeks following treatment, but reaccumulated to pre-treatment levels by 7 months old (m.o.). Vector genome DNA decreased between 3 days and 3 weeks in the liver following vector administration, mainly through the loss of single-stranded genomes; however, double-stranded vector genomes were more stable. Although CD8+ lymphocytic infiltrates were present in the liver, partial biochemical correction was sustained at 7 m.o. The development of efficacious AAV vector-mediated gene therapy could significantly reduce the impact of long-term complications in GSD-Ia, including hypoglycemia, hyperlipidemia and growth failure.     

Immune responses to adenovirus and adeno-associated virus in humans.

Vectors based on human adenovirus (Ad) and adeno-associated virus (AAV) are being evaluated for human gene therapy. The response of the host to the vector, in terms of antigen-specific immunity, will play a substantial role in clinical outcome. We have surveyed cohorts of normal subjects and cystic fibrosis patients for pre-existing immunity to these viruses, caused by naturally acquired infections. A number of humoral and cellular assays to adenovirus serotype 5 (Ad5) and adeno-associated virus serotype 2 (AAV2) were performed from serum and peripheral blood mononuclear cells. Virtually all subjects had Ig to Ad5 although only 55% of these antibodies neutralized virus (NAB). Approximately two of three patients demonstrated CD4+ T cells that proliferated to Ad antigens of which most were of the TH1 subset, based on cytokine secretion. A substantially different pattern of immune responses was observed to AAV2. Although virtually all patients had Ig to AAV2, most of these antibodies were not neutralizing (32% NAB) and only 5% of patients had peripheral blood lymphocytes that proliferated in response to AAV2 antigens. These studies demonstrate marked heterogeneity in pre-existing immunity to Ad5 and AAV2 in human populations. The impact of these findings on outcome following gene therapy will require further study.     

Recombinant adenovirus expressing adeno-associated virus cap and rep proteins supports production of high-titer recombinant adeno-associated virus.

It has been difficult to produce a chimeric vector containing both Ad and AAV rep and cap, and to grow such chimeric vectors in 293 cells. By recombination in vitro in a bacterial host, we were able to produce recombinant plasmid AdAAV (pAdAAVrep-cap), which could be used to generate recombinant AdAAV (rAdAAVrep-cap) after transfection into 293 cells. A recombinant adenovirus, rAdAAVGFP, in which the green fluorescent protein (GFP) gene is flanked by the AAV terminal repeats cloned into the E1-deleted site of Ad was also generated. Co-infection of rAdAAVrep-cap together with rAdAAVGFP into 293 cells resulted in production of high titers of rAAV expressing GFP. It was noted that the titer of rAdAAVrep-cap was lower than the titer of control AdCMVLacZ. The lower titer of rAdAAvrep-cap was associated with expression of Rep protein. Non-homologous recombination occurs after high passage and results in deletions within the AAV rep genes. These results indicate that (1) rAdAAVrep-cap can be produced; (2) rAdAAVrep-cap + rAdAAVGFP is a convenient and efficient way to transfect 293 cells to grow high titer rAAV; and (3) frozen stock is required to avoid propagation of rep-deleted pAdAAVrep-cap.     

Interleukin-10 expression induced by adeno-associated virus vector suppresses proteinuria in Zucker obese rats

Varying degrees of metabolic abnormalities mediated by chronic inflammation are implicated in the chronic glomerular injuries associated with obesity. Interleukin (IL)-10, a pleiotropic cytokine, exerts anti-inflammatory effects in numerous biological settings. In the present study, we explored the biological benefits of adeno-associated virus (AAV) vector-mediated sustained IL-10 expression against the pathological renal characteristics observed in Zucker fatty rats (ZFRs). We injected an AAV vector, encoding rat IL-10 or enhanced green fluorescent protein (GFP) into male ZFRs at 5 weeks of age. Subsequently, the renal pathophysiological changes were analyzed. Persistent IL-10 expression significantly reduced the urinary protein excretion of ZFRs compared with GFP expression (47.1±11.6?mg per mg·creatinine versus 88.8±30.0?mg per mg·creatinine, P<0.01). The serum levels of IL-10 negatively correlated with the urinary protein in AAV-treated rats (r=-0.78, P<0.01). Renal hypertrophy, increased widths in the glomerular basement membrane, and the lack of uniformity and regularity of the foot process of the visceral glomerular epithelial cells of ZFRs were significantly blunted by IL-10 expression. IL-10 also abrogated the downregulation of glomerular nephrin observed in ZFRs treated with the GFP vector. Our findings provide insights into the potential benefit of the anti-inflammatory effects of IL-10 on the overall management of glomerulopathy induced by the metabolic disorders associated with obesity.     

Biochemical and functional evaluation of the sympathectomy produced by the administration of guanethidine to newborn rats.

The administration of guanethidine to newborn rats has been shown by morphological criteria to destroy sympathetic neurons. Newborn rats were injected with guanethidine (50-100 mg/kg/day for 20 days). Upon maturation (at 10 weeks old), the degree of destruction of the sympathetic nervous system (sympathectomy) was assessed. Marked decreases (80-98%) in the norepinephrine concentration in several tissues (heart, spleen, intestine, mesentery, kidney, uterus, vas deferens) were observed in the guanethidine-treated rats when compared to saline-treated controls. No changes were observed in the epinephrine concentration in the adrenals or in the norepinephrine levels in whole brain. Analysis of brain areas showed no change in the norepinephrine levels in brain stem and cerebrum and a small (18%) decrease in the cerebellum. Stimulation of the sympathetic vasomotor outflow in the pithed rat preparation produced almost no response in guanethidine-treated animals. Periarterial nerve stimulation of the isolated perfused kidney preparation also produced essentially no response in guanethidine-treated animals. Isolated intestinal preparations from guanethidine-treated animals responded to nerve stimulation with contractions rather than relaxation as seen in preparations from control animals. Isolated vas deferens preparations responded normally to nerve stimulation despite a 95% decrease in tissue norepinephrine concentration. These data indicate that administration of guanethidine to newborn rats produces a more complete peripheral sympathectomy, especially of the vasculature, than immunosympathectomy or neonatal administration of 6-hydroxydopamine and does so with no significant effect on central noradrenergic neurons.     

小鼠脂联素重组腺相关病毒载体的构建

目的：构建脂联素重组腺相关病毒载体，为进一步研究脂联素的功能提供基础。方法：实验于2003年在中山大学附属第二医院林百欣医学研究中心进行。以带有小鼠脂联素基因的质粒peDNA3．0-Ad为模板，聚合酶链反应克隆脂联素基因，并将聚合酶链反应扩增产物定向克隆于pAAV-MCS载体，重组AAV-MCS-Ad质粒经双酶切和测序鉴定。随后采用磷酸钙转染法将pAAV-MCS-Ad、pAAV-RC，pAAV-Helper质粒共转染AAV-293细胞，包装得到重组腺相关病毒（rAAV-Ad）。回收病毒原液，采用PEG8000和氯仿纯化浓缩病毒。SDS-PAGE及透射电镜鉴定纯化的病毒，并采用Southern blot测定重组腺相关病毒的滴度。Western blotting检测rAAV-Ad转染H4IIE细胞后脂联素蛋白的表达。 结果：①重组质粒AAV-MCS-Ad经双酶切和测序鉴定证实将脂联素基因已正确插入。②SDS-PAGE和电镜均证实rAAV-Ad病毒已构建成功。③Southern blotting测定病毒滴度约为约2．5&;#215;10^14L^-1，通过rAAV-LacZ转染细胞后X-gal染色计数，病毒的感染滴度在（0．83～1．2）&;#215;10^10转导单位／mL。（少Western blotting证实rAAV-Ad转染H4IIE细胞后在30ku处见特异的阳性蛋白条带，证明能有效表达脂联素蛋白。 结论：成功构建了携带小鼠脂联素基因的rAAV-Ad载体，经纯化浓缩后具有较好的纯度和较高滴度，能有效转染H4IIE细胞，并表达脂联素蛋白，具有良好的功能，为进一步研究其茁真核细胞中的功能奠定了基础。     

Delayed expression of adeno-associated virus vector DNA

Two previous reports indicated that recombinant adeno-associated virus (rAAV) vectors were dependent on helper adenovirus (Ad) for efficient conversion of single-stranded (ss) rAAV DNA to the double-stranded (ds) form. This finding is somewhat paradoxical, however, since during a latent infection wild-type (wt)-AAV is rapidly converted to a ds form in the absence of Ad. Our hypothesis was that the effect observed in the previous studies was due to kinetic factors, i.e. to a relative delay in conversion to ds-DNA rather than to an absolute requirement for Ad. To test this, Hela cells were infected with a rAAV-CMV-green fluorescent protein (GFP) vector either in the presence or absence of Ad. Within the first 2 days, Ad infection resulted in a 4-fold increase in AAV vector expression and an augmentation of conversion to a ds-AAV DNA. By 6 days, however, the total number of GFP-expressing cells in the Ad-free culture had exceeded the original number in the Ad co-infected cells, and the conversion to ds-DNA episomes was substantial and ongoing.     

Intracellular transport of recombinant adeno-associated virus vectors

Recombinant adeno-associated viral vectors (rAAVs) have been widely used for gene delivery in animal models, and are currently evaluated for human gene therapy after successful clinical trials in the treatment of inherited, degenerative or acquired diseases, such as Leber congenital amaurosis, Parkinson disease or heart failure. However, limitations in vector tropism, such as limited tissue specificity and insufficient transduction efficiencies of particular tissues and cell types, still preclude therapeutic applications in certain tissues. Wild-type adeno-associated viruses (AAVs) are defective viruses that require the presence of a helper virus to complete their life cycle. On the one hand, this unique property makes AAV vectors one of the safest available viral vectors for gene delivery. On the other, it also represents a potential obstacle because rAAV vectors have to overcome several biological barriers in the absence of a helper virus to transduce successfully a cell. Consequently, a better understanding of the cellular roadblocks that limit rAAV gene delivery is crucial and, during the last 15 years, numerous studies resulted in an expanding body of knowledge of the intracellular trafficking pathways of rAAV vectors. This review describes our current understanding of the mechanisms involved in rAAV attachment to target cells, endocytosis, intracellular trafficking, capsid processing, nuclear import and genome release with an emphasis on the most recent discoveries in the field and the emerging strategies used to improve the efficiency of AAV-derived vectors.