Muscle injection of rAAV/mFIX to secrete clotting factor IX corrects the hemorrhagic tendencies in hemophilia B mice

Recombinant AAV particles of high titer (>1013 virus genome/mL) were prepared according to the rHSV/AAV helper virus method. After intramuscular injection of viral vectors in the hind limb, a sustained elevated level (>370 ng/mL) of murine FIX expression in the plasma of hemophilia B mouse was detected and persisted for more than 350 days. The biological activity reached 30% of normal levels, and bleeding symptoms in the treated mice were significantly alleviated. No anti-FIX antibody (inhibitor) was detected, though anti-AAV antibodies were found at a very low level after single injection. Repeated injection with rAAV/mFIX led to a variation in anti-AAV antibody levels between the two groups which had received different doses. Results from tissue analysis confirmed the skeletal muscle as the origin for circulating functional mFIX. Our results suggest that AAV-mediated gene transfer offers a promising method of gene therapy for hemophilia B.     

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 IX (hFIX) gene in skeletal muscle of coagulation factor IX deficiency mouse strain (FactorIX-knockout) is evaluated. The muscle creatine kinase enhancer (MCK) and β-actin promoter (βA) were used to drive the hFIX minigene (hFIXml), which was flanked by AAV inverted terminal repeats (ITRs). Following intramuscular injection of high titer (2.5 × 10¹¹ vector genomes/mL) of rAAV, 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-mediated gene transfer and skeletal muscle-specific expression of hFIX is a feasible strategy for treating patients for hemophilia B.     

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.     

Heparin binding directs activation of T cells against adeno-associated virus serotype 2 capsid

Activation of T cells to the capsid of adeno-associated virus (AAV) serotype 2 vectors has been implicated in liver toxicity in a recent human gene therapy trial of hemophilia B. To further investigate this kind of toxicity, we evaluated T-cell responses to AAV capsids after intramuscular injection of vectors into mice and nonhuman primates. High levels of T cells specific to capsids of vectors based on AAV2 and a phylogenetically related AAV variant were detected. Vectors from other AAV clades such as AAV8 (ref. 3), however, did not lead to activation of capsid-specific T cells. Through the generation of AAV2-AAV8 hybrids and the creation of site-directed mutations, we mapped the domain that directs the activation of T cells to the RXXR motif on VP3, which was previously shown to confer binding of the virion to heparan sulfate proteoglycan (HSPG). Evaluation of natural and engineered AAV variants showed direct correlations between heparin binding, uptake into human dendritic cells (DCs) and activation of capsid-specific T cells. The role of heparin binding in the activation of CD8(+) T cells may be useful in modulating the immunogenicity of antigens and improving the safety profile of existing AAV vectors for gene therapy.     

Repeated Delivery of Adeno-Associated Virus Vectors to the Rabbit Airway

Efficient local expression from recombinant adeno-associated virus (rAAV)-cystic fibrosis (CF) transmembrane conductance regulator (CFTR) vectors has been observed in the airways of rabbits and monkeys for up to 6 months following a single bronchoscopic delivery. However, it is likely that repeated administrations of rAAV vectors will be necessary for sustained correction of the CF defect in the airways. The current study was designed to test the feasibility of repeated airway delivery of rAAV vectors in the rabbit lung. After two doses of rAAV-CFTR to the airways, rabbits generated high titers of serum anti-AAV neutralizing antibodies. Rabbits then received a third dose of a rAAV vector containing the green fluorescent protein (GFP) reporter gene packaged in either AAV serotype 2 (AAV2) or serotype 3 (AAV3) capsids. Each dose consisted of 1 ml containing 5 x 10(9) DNase-resistant particles of rAAV vector, having no detectable replication-competent AAV or adenovirus. Three weeks later, GFP expression was observed in airway epithelial cells despite high anti-AAV neutralizing titers at the time of delivery. There was no significant difference in the efficiency of DNA transfer or expression between the rAAV3 and rAAV2 groups. No significant inflammatory responses to either repeated airway exposure to rAAV2-CFTR vectors or to GFP expression were observed. These experiments demonstrate that serum anti-AAV neutralizing antibody titers do not predict airway neutralization in vivo and that repeated airway delivery rAAV allows for safe and effective gene transfer.     

比较不同血清型AAV携带HBV基因组建立乙肝小鼠模型效果

近年来,用8型腺相关病毒携带1.3拷贝HBV(Hepatitis B virus)基因组建立的HBV持续感染小鼠模型受到越来越多的关注。本研究比较了除AAV8之外的其他4种血清型重组腺相关病毒(Recombinant adeno-associated virus,rAAV)建立乙肝小鼠模型效果。首先,将携带1.3拷贝ayw亚型HBV基因组的1型、2型、5型、8型、9型腺相关病毒分别以1×10~(11) vg/只(Viral genome,vg)的剂量尾静脉注射C57BL/6J小鼠;利用ELISA方法监测小鼠血清中HBeAg和HBsAg表达水平;用定量PCR方法检测小鼠血清和肝脏中HBV DNA拷贝数;用免疫组化方法检测小鼠肝脏中HBc Ag的表达;用HE染色检测小鼠肝脏病理变化。结果显示,在持续8周中,5组小鼠血清中都检测到HBeAg和HBsAg的表达,血清和肝脏中均检测到HBV DNA的存在。HBeAg、HBsAg、HBV DNA表达水平高低依次为AAV8AAV9AAV1AAV5AAV2。5组小鼠用免疫组化方法都检测到肝脏中HBcAg表达,HE染色病理检测均观察到不同程度的肝损伤。本研究扩大了能用于建立乙肝小鼠持续感染模型可选择的AAV载体种类,发现虽然AAV1、2、5、9的建模效果不如AAV8,但它们都可以介导建立持续感染的乙肝小鼠模型,建模效果依次为AAV8AAV9AAV1AAV5AAV2。其中AAV9介导的建模效果与AAV8载体最为接近,可以替代AAV8载体用于有效地建立HBV持续感染的小鼠模型。     

Encapsidation of adeno-associated virus type 2 Rep proteins in wild-type and recombinant progeny virions: Rep-mediated growth inhibition of primary human cells.

The adeno-associated virus type 2 (AAV) arrests the growth of primary human fibroblasts in vitro at high particle-to-cell ratios. To test the role of AAV gene expression in the observed growth inhibition, primary human cells were infected, under identical conditions, with wild-type (wt) AAV or with recombinant AAV that lacked all viral promoters and coding sequences. Significant, dose-dependent growth inhibition of primary human cells was observed with both wt and recombinant AAV at particle-to-cell ratios equal to or exceeding 10(4). In contrast, neither virus affected the growth of immortalized human cells even at a 10-fold-higher particle-to-cell ratio. AAV-induced growth arrest could be overcome by reculturing cells after treatment with trypsin. Even after reculturing, cells still harbored the proviral AAV genome. Thus, neither integration nor expression of the AAV genome appears to be required for the virus-induced growth-inhibitory effect on primary human cells. The growth-inhibitory effect of AAV was hypothesized to be mediated by virion-associated AAV Rep proteins, since these proteins have been reported to inhibit cellular DNA synthesis. Rep proteins tightly associated with wt as well as recombinant AAV could be detected on Western blots. Coinfection by adenovirus was necessary and sufficient for ample replication of recombinant AAV genomes lacking the rep gene. Although wt AAV-like particles arose during production of the recombinant AAV stocks, their low-titer levels were insufficient to cause the observed growth inhibition. AAV rep gene expression from these contaminating particles was not required for replication of the recombinant AAV genomes, which could be detected even in the absence of de novo Rep protein synthesis. Exposure of recombinant AAV to anti-AAV Rep protein antibodies did not abrogate viral infectivity. These results suggest that biologically active Rep proteins are encapsidated in mature progeny AAV particles. AAV Rep protein-mediated growth inhibition of primary human cells has implications in the use of AAV-based vectors in human gene therapy.     

Secretion of a TNFR:Fc fusion protein following pulmonary administration of pseudotyped adeno-associated virus vectors

This study evaluated and compared delivery of the tumor necrosis factor alpha receptor (TNFR)-immunoglobulin G1 (IgG1) Fc fusion (TNFR:Fc) gene to the lung by single and repeat administrations of multiple pseudotyped adeno-associated virus (AAV) vectors as a means for achieving systemic distribution of the soluble TNFR:Fc protein. A single endotracheal administration of AAV[2/5]cytomegalovirus (CMV)-TNFR:Fc vector (containing the AAV2 inverted terminal repeats and AAV5 capsid) to the rat lung resulted in long-term, high levels of serum TNFR:Fc protein that gradually declined over a period of 8 months. Endotracheal delivery of AAV[2/1]CMV-TNFR:Fc resulted in serum TNFR:Fc protein levels that were detectable for at least 4 months but were 10-fold lower than that of the AAV[2/5] vector. In contrast, secretion of the TNFR:Fc protein following pulmonary delivery of AAV[2/2]CMV-TNFR:Fc vector was very inefficient, and the protein was detected in the blood only when an airway epithelial cell-specific promoter, CC10, was substituted for the CMV enhancer/promoter to control transgene expression. In the context of AAV[2/5], the CC10 promoter was as efficient as CMV enhancer/promoter in generating similar levels of systemic TNFR:Fc protein, suggesting that this protein is secreted primarily from the airway epithelium. In mice, comparable long-term secretion of TNFR:Fc protein was demonstrated after AAV[2/2] and AAV[2/5] delivery, although the kinetics of transduction appeared to be different. All pseudotyped AAV vectors elicited serum anti-AAV capsid-neutralizing antibody responses, but these did not prevent lung transduction and efficient secretion of TNFR:Fc protein to the circulation following readministration with AAV[2/5]. These results highlight the potential utility of AAV vectors containing serotype 5 capsid to deliver and redeliver genes of secreted proteins to the lung to achieve long-term systemic protein expression.     

Latent Adeno-Associated Virus Infection Elicits Humoral but Not Cell-Mediated Immune Responses in a Nonhuman Primate Model

Latent infection with wild-type (wt) adeno-associated virus (AAV) was studied in rhesus macaques, a species that is a natural host for AAV and that has some homology to humans with respect to the preferred locus for wt AAV integration. Each of eight animals was infected with an inoculum of 10(10) IU of wt AAV, administered by either the intranasal, intramuscular, or intravenous route. Two additional animals were infected intranasally with wt AAV and a helper adenovirus (Ad), while one additional animal was inoculated with saline intranasally as a control. There were no detectable clinical or histopathologic responses to wt AAV administration. Molecular analyses, including Southern blot, PCR, and fluorescence in situ hybridization, were performed 21 days after infection. These studies indicated that AAV DNA sequences persisted at the sites of administration, albeit at low copy number, and in peripheral blood mononuclear cells. Site-specific integration into the AAVS1-like locus was observed in a subset of animals. All animals, except those infected by the intranasal route with wt AAV alone, developed a humoral immune response to wt AAV capsid proteins, as evidenced by a >/=fourfold rise in anti-AAV neutralizing titers. However, only animals infected with both wt AAV and Ad developed cell-mediated immune responses to AAV capsid proteins. These findings provide some insights into the nature of anti-AAV immune responses that may be useful in interpreting results of future AAV-based gene transfer studies.     

Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A, a high catalytic activity mutation of human coagulation factor IX

A mutant human factor IX with arginine at 338 residual changed to alanine (hFIXR338A) by site-directed mutagenesis was introduced into AAV vectors, and a recombinant adeno-associ-ated viral vector containing hFIXR338A, prepared by rHSV/AAV hybrid helper virus system, was directly introduced to the hind leg muscle of factor IX knock out mice. The expression and the biological activity of human factor IX mutant, hFIXR338A, and the immune response against it in the treated mice were assayed and detected. The results showed that (i) the high-level expression of human factor IX mutant protein, hFIXR338A, has been detected in rAAV-hFIXR338A treated hemophilia B mice and lasted more than 15 weeks; (ii) the clotting activity of hFIXR338A in plasma is 34.2%± 5.23%, which is remarkably higher than that of (14.27%±3.4%) of wild type hFIX treated mice in the activated partial thromboplastin assay; (iii) immune response against factor IX R338A was absent, with no factor IX mutant protein (hFIXR338A) inhibitors deve     

Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A, a high catalytic activity mutation of human coagulation factor IX

A mutant human factor IX with arginine at 338 residual changed to alanine (hFIXR338A) by site-directed mutagenesis was introduced into AAV vectors, and a recombinant adeno-associ- ated viral vector containing hFIXR338A, prepared by rHSV/AAV hybrid helper virus system, was directly introduced to the hind leg muscle of factor IX knock out mice. The expression and the biological activity of human factor IX mutant, hFIXR338A, and the immune response against it in the treated mice were assayed and detected. The results showed that (i) the high-level expression of human factor IX mutant protein, hFIXR338A, has been detected in rAAV-hFIXR338A treated hemophilia B mice and lasted more than 15 weeks; (ii) the clotting activity of hFIXR338A in plasma is 34.2%± 5.23%, which is remarkably higher than that of (14.27% ± 3.4%) of wild type hFIX treated mice in the activated partial thromboplastin assay; (iii) immune response against factor IX R338A was absent, with no factor IX mutant protein (hFIXR338A) inhibitors development in the treated mice; and (iv) no local or systemic side-effects and toxicity associated with the gene transfer were found. It demonstrated the potential use of treating hemophilia B by recombinant adeno-associated viral vectors with mutant hFIXR338A gene, an alternative strategy for hemophilia B gene therapy to wild-type human factor IX.     

Epitope mapping of human anti-adeno-associated virus type 2 neutralizing antibodies: implications for gene therapy and virus structure

Recombinant adeno-associated virus type 2 (AAV) is a common vector used in human gene therapy protocols. We characterized the humoral immune response to AAV and observed that 80% of normal human subjects have anti-AAV antibodies and that 18% have neutralizing antibodies. To analyze the effect of neutralizing antibodies on AAV readministration, we attempted to deliver recombinant AAV expressing human factor IX (AAV-hFIX) intraportally into the livers of mice which had been preexposed to AAV and shown to harbor a neutralizing antibody response. While all naive control mice expressed hFIX following administration of AAV-hFIX, none of the mice with preexisting immunity expressed hFIX, even after transient immunosuppression at the time of the second administration with anti-CD4 or anti-CD40L antibodies. This suggests that preexisting immunity to AAV, as measured by a neutralizing antibody response, may limit AAV-mediated gene delivery. Using human sera in an enzyme-linked immunosorbent assay for AAV and a capsid peptide scan library to block antibody binding, we mapped seven regions of the AAV capsid containing immunogenic epitopes. Using pools of these peptides to inhibit the binding of neutralizing antibodies, we have identified a subset of six peptides which potentially reconstitute a single neutralizing epitope. This information may allow the design of reverse genetic approaches to circumvent the preexisting immunity that can be encountered in some individuals.     

Humoral immune responses to AAV gene therapy in the ocular compartment

Viral vectors can be utilised to deliver therapeutic genes to diseased cells. Adeno-associated virus (AAV) is a commonly used viral vector that is favoured for its ability to infect a wide range of tissues whilst displaying limited toxicity and immunogenicity. Most humans harbour anti-AAV neutralising antibodies (NAbs) due to subclinical infections by wild-type virus during infancy and these pre-existing NAbs can limit the efficiency of gene transfer depending on the target cell type, route of administration and choice of serotype. Vector administration can also result in de novo NAb synthesis that could limit the opportunity for repeated gene transfer to diseased sites. A number of strategies have been described in preclinical models that could circumvent NAb responses in humans, however, the successful translation of these innovations into the clinical arena has been limited. Here, we provide a comprehensive review of the humoral immune response to AAV gene therapy in the ocular compartment. We cover basic AAV biology and clinical application, the role of pre-existing and induced NAbs, and possible approaches to overcoming antibody responses. We conclude with a framework for a comprehensive strategy for circumventing humoral immune responses to AAV in the future.     

A comparative study of hFIX expression mediated by rAAV8 and rAAV1 administrated intramuscularly

BACKGROUND: Recombinant AAV serotype 8 (rAAV8) vector is relatively new for gene therapy. In this study, the hFIX expression mediated by rAAV8 injected intramuscularly was compared with that by rAAV1. METHODS: rAAV8-hFIX or rAAV1-hFIX viruses were injected intramuscularly into two hind limbs of mice at doses of 5x10(10) gc and 2.5x10(12) gc (genome copy). The hFIX expression in the mouse plasma was detected by ELISA, APTT and Western blotting. The virus distribution was analyzed by immunohistochemical assay. RESULTS: When the mice were infected with 5x10(10) gc virus, high levels of hFIX in the plasma of five rAAV8-hFIX virus-infected mice were detected 2 weeks after injection. A hFIX peak above 5000 ng/mL appeared between 2 and 6 weeks after injection. Relatively low levels of hFIX were detected in the plasma of rAAV1-hFIX virus-infected mice 2 weeks after injection. An hFIX peak above 3000 ng/mL appeared between 4 and 10 weeks after injection. However, much lower levels of hFIX were detected in mice infected with higher dose of rAAV8 virus. The hFIX in the mouse plasma was active biologically. The viruses were distributed mainly in the muscles of hind limbs. DISCUSSION: Gene expression mediated by rAAV8 was sooner and stronger than that by rAAV1 after intramuscular administration. Inhibition might have been triggered markedly by rAAV8 at high doses.     

Directed evolution of adeno-associated virus yields enhanced gene delivery vectors

Adeno-associated viral vectors are highly safe and efficient gene delivery vehicles. However, numerous challenges in vector design remain, including neutralizing antibody responses, tissue transport and infection of resistant cell types. Changes must be made to the viral capsid to overcome these problems; however, very often insufficient information is available for rational design of improvements. We therefore applied a directed evolution approach involving the generation of large mutant capsid libraries and selection of adeno-associated virus (AAV) 2 variants with enhanced properties. High-throughput selection processes were designed to isolate mutants within the library with altered affinities for heparin or the ability to evade antibody neutralization and deliver genes more efficiently than wild-type capsid in the presence of anti-AAV serum. This approach, which can be extended to additional gene delivery challenges and serotypes, directs viral evolution to generate 'designer' gene delivery vectors with specified, enhanced properties.     

BiP表达下调改善双载体转断裂FVIII基因的功效

双载体转凝血Ⅷ因子（FⅧ）基因可作为一种转基因策略克服腺相关病毒(AAV)载体容量限制,但重链分泌的低效性影响转基因功效. 为提高重链分泌,本文用RNA干扰技术下调内质网内蛋白伴侣分子免疫球蛋白重链结合蛋白(BiP)的表达,观察对HEK293细胞双载体共转FⅧ基因分泌重链和生物活性的影响. 结果显示,RNA干扰可明显下调BiP表达,但不影响细胞生长; ELISA检测BiP下调细胞单独转重链基因时的重链分泌量为98±38 ng/mL,与轻链共转基因时显著升高到157±32 ng/mL,明显高于对照细胞单独转重链基因和共转重链和轻链基因的重链分泌量(分别为29±8 ng/mL和79±19 ng/mL);Cotest法检测显示,BiP下调细胞共转重链和轻链基因细胞分泌的凝血生物活性为0.73±0.23 IU/mL,明显高于对照细胞共转重链和轻链基因(0.39±0.07 IU/mL). 结果表明, BiP表达下调通过促进重链分泌,可提高双载体共转FⅧ基因的功效,为进一步动物体内双AAV载体转FⅧ基因的甲型血友病基因治疗研究提供了实验依据.     

Gene therapy using AAV

AAV (adeno-associated virus) vectors are considered to be promising gene-delivery vehicles for gene therapy, because they are derived from non-pathogenic virus, efficiently transduce non-dividing cells, and cause long-term gene expression. Appropriate AAV serotypes are utilized depending on the type of target cells. Among various neurological disorders, Parkinson's disease (PD) is one of the most promising candidates of gene therapy. PD is a progressive neurodegenerative disorder that predominantly affects dopaminergic neurons in the substantia nigra. One of the major approaches to gene therapy of PD is the intrastriatal expression of dopamine (DA)-synthesizing enzyme genes. As for the initial step of clinical application, AAV vector-mediated AADC (aromatic L-amino acid decarboxylase; the enzyme converting L-DOPA to DA) gene transfer in combination with oral administration of L-DOPA would be appropriate, since DA production can be regulated by adjusting the dose of L-DOPA. Second, intramuscular injection of AAV vectors is appropriate to protein-supplement gene therapy. Monogenic diseases such as hemophilia and Fabry disease are suitable candidates. Regarding cancer gene therapy, AAV vectors may be utilized to inhibit tumor angiogenesis, metastasis, and invasion. When long-term transgene expression in stem cells is needed, a therapeutic gene should be introduced with a minimal risk of insertional mutagenesis. To this end, site-specific integration into the AAVS1 locus on the chromosome 19 (19q13.4) by using the integration machinery of AAV would be particularly valuable.     

The treatment of hemophilia A: from protein replacement to AAV-mediated gene therapy

Factor VIII (FVIII) is an essential component in blood coagulation, a deficiency of which causes the serious bleeding disorder hemophilia A. Recently, with the development of purification level and recombinant techniques, protein replacement treatment to hemophiliacs is relatively safe and can prolong their life expectancy. However, because of the possibility of unknown contaminants in plasma-derived FVIII and recombinant FVIII, and high cost for hemophiliacs to use these products, gene therapy for hemophilia A is an attractive alternative to protein replacement therapy. Thus far, the adeno-associated virus (AAV) is a promising vector for gene therapy. Further improvement of the virus for clinical application depends on better understanding of the molecular structure and fate of the vector genome. It is likely that hemophilia will be the first genetic disease to be cured by somatic cell gene therapy.     

Productive replication of adeno-associated virus can occur in human papillomavirus type 16 (HPV-16) episome-containing keratinocytes and is augmented by the HPV-16 E2 protein

We used a sensitive assay to test whether an adeno-associated virus (AAV) productive replication cycle can occur in immortalized human keratinocytes carrying episomal human papillomavirus type 16 (HPV-16) DNA. Following transfection with cloned AAV DNA, infectious AAV was produced, and the infectivity was blocked by anti-AAV antiserum. The HPV-16 E2 protein substantially increased the yield of AAV. Other HPV early proteins did not, in our experiments, show this ability. E2 has been shown to be able to affect p53 levels and to block cell cycle progression at mitosis. We tested the effect of changes in p53 expression on AAV replication and found that large differences in the level of p53 did not alter AAV DNA replication. In extension of this, we found that cellular help for AAV in response to stress was also independent of p53. To test if a mitotic block could trigger AAV DNA replication, we treated the cells with the mitotic inhibitor nocodazole. AAV DNA replication was stimulated by the presence of nocodazole in these and a number of other cell types tested. Yields of infectious virus, however, were not increased by this treatment. We conclude that the HPV-16 E2 protein stimulates AAV multiplication in these cells and propose that this occurs independently of the effects of E2 on p53 and cell cycle progression. Since the effect of E2 was not seen in keratinocytes lacking the HPV-16 episome, we suggest that E2 can help AAV by working in concert with other HPV-16 proteins.