Induction of dystrophin expression by exon skipping in mdx mice following intramuscular injection of antisense oligonucleotides complexed with PEG-PEI copolymers.

Antisense oligonucleotides (AOs) with 2-O-methyl modifications can circumvent dystrophin mutations via exon skipping and, it is hoped, can become drugs for treatment of Duchenne muscular dystrophy (DMD). However, AO-based approaches are hindered by a lack of effective carriers to facilitate delivery of AOs to myonuclei. We examined whether copolymers composed of cationic poly(ethylene imine) (PEI) and polyethylene glycol (PEG) can enhance AO transfection in skeletal muscle of mdx mice. Single intramuscular injections of AO complexed with low Mw PEI2000(PEG550) copolymers into TA muscles of mdx mice resulted in widespread distribution of dystrophin-positive fibers at 3 weeks after injection, with no apparent cytotoxicity. Overall, injections of these low Mw polyplexes, which formed 250-nm aggregate particles, resulted in about sixfold more dystrophin-positive fibers than AO alone. Western analysis confirmed the dystrophin expression in these muscles. Surprisingly, injections of AO complexed with high Mw PEI25000(PEG5000) copolymers, which formed smaller nonaggregated particles, produced about threefold fewer dystrophin-positive fibers than injections of the low Mw polyplexes. We conclude that low Mw PEI2000(PEG550) copolymers function as high-capacity, nontoxic AO carriers suitable for in vivo transfection of skeletal muscle and are promising compounds for potential use in molecular therapy of DMD.     

Immune rejection of human dystrophin following intramuscular injections of naked DNA in mdx mice

Intramuscular administration of plasmid expressing full-length human dystrophin in dystrophin-deficient adult mdx mice resulted in humoral and weak specific T cell responses against the human dystrophin protein. Following plasmid injection, human dystrophin was detected in the injected muscles at 7 days, but decreased thereafter. Anti-dystrophin antibodies were found 21 days following plasmid injection, which coincided with transient myositis. This immune rejection prevented the mice from expressing human dystrophin after a second plasmid injection. No anti-DNA antibodies were found. Anti-dystrophin antibodies were seen in a smaller proportion of plasmid-injected dystrophin-competent C57BL/10 mice, suggesting that the immune rejection of dystrophin may be explained partially by species differences in the dystrophin protein.     

Restoration of dystrophin expression in mdx mice by intravascular injection of naked DNA containing full-length dystrophin cDNA

Duchenne muscular dystrophy (DMD) is a lethal, X-linked, recessive disease caused by a defect in the dystrophin gene. No effective therapy is available. Dystrophin gene transfer to skeletal muscle has been proposed as a treatment for DMD. However, successful treatment for DMD requires restoration of dystrophin in the affected muscle fibers to at least 20% of the normal level. Current gene transfer methods such as intramuscular injection of viral vector or naked DNA can only transfect a small area of muscle, and therefore is of little clinical utility. We have developed a semisystemic method for gene transfer into skeletal muscle of mdx mice, an animal model for DMD. Naked DNA was injected through the tail artery or vein of mice, in which the aorta and the vena cava were clamped at the location just below the kidneys. The DNA solution was thus forced into the blood vessels of both legs. Luciferase gene expression was detected in all muscle groups in both legs. The effects of injection speed, injection volume, and ischemia time on gene expression were also optimized. LacZ staining was used to check the spread of gene expression in muscle. Although the percentage of transfected fibers was modest (approximately 10%), beta-galactosidase was found in all muscle groups of both legs. Finally, plasmid DNA encoding full-length dystrophin gene was injected into mdx mice and widespread restoration of dystrophin protein was observed in all muscles of both hind limbs. In conclusion, these results demonstrate that the semisystemic delivery of naked DNA is a potential approach towards the long-term goal of gene therapy for DMD.     

Highly efficient gene transfer into murine liver achieved by intravenous administration of naked Epstein-Barr virus (EBV)-based plasmid vectors

Naked plasmid DNA (pDNA) injection could become an alternative procedure to viral and nonviral gene delivery systems. We have previously shown that Epstein-Barr virus (EBV)-based plasmid vectors containing the EBV nuclear antigen 1 (EBNA1) gene and the oriP sequence enable quite high and long-lasting expression in various in vitro and in vivo transfection systems. The EBV-based plasmids were intravenously injected into mice via their tail vein under high pressure. A large amount of the marker gene product was expressed in the liver; as much as 320 microg of luciferase was demonstrated per gram of liver at 8 to 24 h after a single injection with 10 microg of DNA. More than 70% of liver cells stained with X-gal when beta-gal gene was transferred. The expression level was significantly higher than that obtained by conventional pDNA lacking the EBNA1 gene and oriP. On day 35 after the transfection, the expression from the EBV-based plasmid was approximately 100-fold stronger than the conventional pDNA gene expression. Both the EBNA1 gene and oriP are a prerequisite for the augmentation of the transfection efficiency. These results suggest that the intravascular transfection with naked EBV-based plasmid may provide a quite efficient, simple and convenient means to transduce therapeutic genes in vivo into the liver.     

Long-term doxycycline-regulated transgene expression in the retina of nonhuman primates following subretinal injection of recombinant AAV vectors.

Adeno-associated viral gene therapy has shown promise for the treatment of inherited and acquired retinal disorders. In most applications, regulation of expression is a critical concern for both safety and efficacy. The purpose of our study was to evaluate the ability of the tetracycline-regulatable system to establish long-term transgene regulation in the retina of nonhuman primates. Three rAAV vectors expressing the tetracycline-dependent transactivator (rtTA) under the control of either the ubiquitous CAG promoter or the specific RPE65 promoter (AAV2/5.CAG.TetOn.epo, AAV2/4.CAG.TetOn.epo, and AAV2/4.RPE65.TetOn.epo) were generated and administered subretinally to seven macaques. We demonstrated that repeated inductions of transgene expression in the nonhuman primate retina can be achieved using a Tet-inducible system via rAAV vector administration over a long period (2.5 years). Maximum erythropoietin (EPO) secretion in the anterior chamber depends upon the rAAV serotype and the nature of the promoter driving rtTA expression. We observed that the EPO isoforms produced in the retina differ from one another based on the transduced cell type of origin within the retina and also differ from both the physiological EPO isoforms and the isoforms produced by AAV-transduced skeletal muscle.     

Long-term expression of full-length human dystrophin in transgenic mdx mice expressing internally deleted human dystrophins

One of the possible therapies for Duchenne muscular dystrophy (DMD) is the introduction of a functional copy of the dystrophin gene into the patient. For this approach to be effective, therapeutic levels and long-term expression of the protein need to be achieved. However, immune responses to the newly expressed dystrophin have been predicted, particularly in DMD patients who express no dystrophin or only very truncated versions. In a previous study, we demonstrated a strong humoral and cytotoxic immune response to human dystrophin in the mdx mouse. However, the mdx mouse was tolerant to murine dystrophin, possibly due to the endogenous expression of dystrophin in revertant fibres or the other nonmuscle dystrophin isoforms. In the present study, we delivered human and murine dystrophin plasmids by electrotransfer after hyaluronidase pretreatment to increase gene transfer efficiencies. Tolerance to murine dystrophin was still seen with this improved gene delivery. Tolerance to exogenous recombinant full-length human dystrophin was seen in mdx transgenic lines expressing internally deleted versions of human dystrophin. These results suggest that the presence of revertant fibres may prevent the development of serious immune responses in patients undergoing dystrophin gene therapy.     

Neonatal intramuscular injection with recombinant adeno-associated virus results in prolonged beta-glucuronidase expression in situ and correction of liver pathology in mucopolysaccharidosis type VII mice

For many metabolic diseases, early correction of the inherited deficiency is required to prevent long-term sequelae. We examined the ability of adeno-associated virus (AAV) to mediate efficient gene transfer during the neonatal period in mice with the lysosomal storage disease mucopolysaccharidosis type VII (MPS VII). Quadriceps of newborn MPS VII mice were injected with an AAV vector containing human beta-glucuronidase (GUSB) cDNA. High-level intramuscular GUSB expression was seen as early as 2 weeks of age, and persisted for at least 16 weeks with no reduction in activity. In addition, GUSB activity was detected in both liver and spleen at later time points. The level of GUSB activity resulted in a significant reduction in lysosomal storage in the liver and a minimal reduction in the spleen at 16 weeks. However, the temporal and spatial pattern of hepatic GUSB activity, coupled with the presence of GUSB cDNA in liver sections, suggests that hematogenous dissemination of virus at the time of injection led to gene transfer to hepatic cells. These results demonstrate that AAV vectors can successfully infect neonatal muscle and persist through the rapid growth phase following birth. However, GUSB secretion from an intramuscular source is inefficient, limiting the therapeutic efficacy of this approach.     

NC37-R1 Epstein-Barr virus (EBV): a possible recombinant between intracellular NC37 viral DNA and superinfecting P3HR-1 EBV

The NC37-R1 cell line, established after transformation of human cord blood lymphocytes with Epstein-Barr virus (EBV) recovered from P3HR-1 superinfected NC37 cells, spontaneously produces viral particles with transforming but without early antigen-inducing properties. Progeny virus of NC37-R1 has retained its biological characteristics of spontaneous virus release and transformation up to four cycles of transformation at present. Analysis of purified NC37-R1 virion DNA, after cleavage with restriction endonuclease Hind III and comparison of its fragments with P3HR-1 EBV as well as intracellular NC37 viral DNA using the blot hybridization technique, suggests that NC37-R1 originates from a recombination between superinfecting P3HR-1 and endogenous NC37 EBV DNA.     

Phase I trial of intramuscular injection of a recombinant adeno-associated virus serotype 2 alphal-antitrypsin (AAT) vector in AAT-deficient adults

A phase I trial of intramuscular injection of a recombinant adeno-associated virus serotype 2 (rAAV2) alpha1-antitrypsin (AAT) vector was performed in 12 AAT-deficient adults, 10 of whom were male. All subjects were either homozygous for the most common AAT mutation (a missense mutation designated PI*Z) or compound heterozygous for PI*Z and another mutation known to cause disease. There were four dose cohorts, ranging from 2.1 x 10(12) vector genomes (VG) to 6.9 x 10(13) VG, with three subjects per cohort. Subjects were injected sequentially in a dose-escalating fashion with a minimum of 14 days between patients. Subjects who had been receiving AAT protein replacement discontinued that therapy 28 days before vector administration. There were no vector-related serious adverse events in any of the 12 participants. Vector DNA sequences were detected in the blood between 1 and 3 days after injection in nearly all patients receiving doses of 6.9 x 10(12) VG or higher. Anti-AAV2 capsid antibodies were present and rose after vector injection, but no other immune responses were detected. One subject who had not been receiving protein replacement exhibited low-level expression of wild-type M-AAT in the serum (82 nM), which was detectable 30 days after receiving an injection of 2.1 x 10(13) VG. Unfortunately, residual but declining M-AAT levels from the washout of the protein replacement elevated background levels sufficiently to obscure any possible vector expression in that range in most of the other individuals in the higher dose cohorts.     

Establishment of specific antibody producing human lines by antigen preselection and Epstein-Barr virus (EBV)-transformation.

Specific antibody producing human cell lines were established by preselecting antigen binding B-lymphocytes and subsequently transforming ("immortalizing") them with Epstein-Barr virus (EBV). NNP-binding B-cells were isolated from the blood of three donors with high anti-NNP titers. EBV-transformation led to polyclonal cell lines that had 15-18% NNP receptor positive cells. A similar fraction of the cells produced NNP-specific plaques in the Cunningham-Szenberg assay. Unconcentrated culture media agglutinated NNP-coupled erythrocytes in up to an 1/2,048 dilution and inactivated NNP-coupled T4 bacteriophage in up to an 1/10,000 dilution. EBV-transformed but not similarly NNP-preselected lines of the same donors were completely negative in the rosette, plaque and antibody secretion tests. Supernatants of the antibody producing lines gave no reaction with the non-coupled erythrocytes or with a number of other hapten coupled controls. Anti-NNP antibodies secreted by all three preselected lines were of the IgM kappa type, which was in contrast to the sera of the donors that contained both IgM and IgG antibodies, with both light chain types. Cloning of one NNP-antibody producing line yielded 9 antibody producers out of 30. The positive clones formed NNP rosettes and plaques with 31-86% of the cells and produced anti-NNP of class IgM, Kappa. The cell culture contained 5-16 micrograms IgM/ml cell culture.     

Autoradiographic detection of Epstein-Barr virus (EBV)-associated early antigen in a variety of EBV DNA-containing lymphoblastoid cell lines previously designated as nonproducers.

By using autoradiography combined with 125I-labeled IgG prepared from human sera containing high anti-EA (early antigen) antibody titers, EA has been detected in a low fraction of cells in a variety of EBV DNA-containing cell lines previously designated as negative for this antigen. These positive cell lines are highly inducible for EA following treatment with iododeoxyuridine (IUDR) and also contain multiple copies of the virus genome on average. The correlation between IUDR inducibility and spontaneous expression of EA, in particular, suggests that the ability to express EA in both situations is interrelated.     

Improved safety and titre of murine leukaemia virus (MLV)-based retroviral vectors

Many retroviral vectors based on murine leukaemia virus (MLV) contain the first 420 nucleotides of the gag gene, as this was reported to increase vector titre by increasing the efficiency of RNA packaging. In this study, deletion of this gag sequence from its original location did not decrease the titre of two retroviral vectors, pBabe puro and MFG-S-. The two vectors could be improved by replacing the gag sequence with a CTE from Mason-Pfizer monkey virus (MPMV). This substitution improved vector titre, while eliminating a region of homology between vector and packaging constructs. Gene Therapy (2000) 7, 300-305.     

造血干细胞移植后单个核细胞EBV-DNA定量监测预测移植后淋巴增殖性疾病

目的 应用定量PCR方法 监测造血干细胞移植(HSCT)后单个核细胞EBV含量,评估其临床效果.方法 51例HSCT患者从预处理阶段开始,采用荧光定量PCR方法 每周1次检测外周s血单个核细胞EBV-DNA拷贝数,分析EBV再活化的影响因素以及EBV-DNA拷贝数与发生淋巴增殖性疾病(PTLD)的相关性.结果 51例患者EBV血症累积发生率为58.8%,HSCT后EBV感染的时间晚于CMV感染[分别为(39.6±23.5)d和(25.0±15.1)d,P＜0.01].Allo-HSCT中HLA不合患者EBV血症的累积发生率显著高于HLA相合患者(分别为93.3%和48.1%,P＜0.01),应用ATG组显著高于无ATG组(分别为92.3%和18.7%,P＜0.01),年龄＜20岁组患者显著高于≥20岁组(分别为100%和53.1%,P＜0.01).30例患者中4例(13.3%)EBV血症发展为EBV-PTLD,均为持续2周以上EBV-DNA＞106拷贝/ml的患者(13例中4例).PTLD患者的中位生存时间为19.5(11～75)d.结论 HSCT后EBV活化比率高,尤其是在HLA不相合供者、应用ATG和年龄＜20岁的患者,有必要常规进行单个核细胞EBV-DNA检测.Allo-HSCT患者在EBV-DNA拷贝数＞106拷贝/ml,尤其是持续2周以上者,易进展为PTLD,应给予抢先治疗.     

Stable therapeutic serum levels of human alpha-1 antitrypsin (AAT) after portal vein injection of recombinant adeno-associated virus (rAAV) vectors

Previous work from our group showed that recombinant adeno-associated virus (rAAV) vectors mediated long-term secretion of therapeutic serum levels of human alpha-1 antitrypsin (hAAT) after a single injection in murine muscle. We hypothesized that hepatocyte transduction could be even more efficient, since these cells represent the natural site of AAT production and secretion. To test this hypothesis, rAAV vectors containing the hAAT cDNA driven by either the human elongation factor 1 alpha promoter, the human cytomegalovirus immediate-early promoter (CMV), or the CMV-chicken beta actin hybrid (CB) promoter were injected into the portal or tail veins of adult C57Bl/6 mice. Potentially therapeutic serum levels of hAAT (600 microg/ml) were achieved after portal vein injection of doses of 4 x 10(9) infectious units (IU), a 10-fold lower dose than that required for similar levels of expression via the i.m. route. Serum levels greater than 1 mg/ml were achieved at doses of 3 x 10(10) IU. Southern blotting of liver DNA revealed the presence of circular episomal vector genomes. Immunostaining showed that transgene expression was scattered throughout the liver parenchyma. Similar results were obtained with a rAAV-CB-green fluorescent protein (GFP) vector. There was no evidence of hepatic toxicity. These data indicate that liver-directed rAAV-based gene therapy is effective in the murine model, and hence might be feasible for treatment of human AAT deficiency.     

Plasmid DNA vaccines: investigation of integration into host cellular DNA following intramuscular injection in mice

The primary safety concern for DNA vaccines is their potential to integrate into the host cell genome. We describe an integration assay based on purification of high-molecular-weight genomic DNA away from free plasmid using gel electrophoresis, such that the genomic DNA can then be assayed for integrated plasmid using a sensitive PCR method. The assay sensitivity was approximately 1 plasmid copy/microg DNA (representing approximately 150,000 diploid cells). Using this assay, we carried out integration studies of three different plasmid DNA vaccines, containing either the influenza hemagglutinin, influenza matrix or HIV gag gene. Six weeks after intramuscular injection, free plasmid was detected in treated muscle at levels ranging from approximately 1,000 to 4,000 copies/microg DNA. At 6 months, the plasmid levels ranged between 200 and 800 copies/microg DNA. Gel purification of genomic DNA revealed that essentially all of the detectable plasmid in treated quadriceps was extrachromosomal. If integration had occurred, the frequency was 相似文献