Masashi Urabe

Insect cells as a factory to produce adeno-associated virus type 2 vectors

Recombinant adeno-associated viruses (rAAV) are produced transiently in mammalian cells usually by cotransfecting two or three plasmids containing AAV genes, adenovirus helper genes, and a vector genome. Expansion and transfection of adherent cells limit the scale of rAAV production. Efficient transfection is performed with cells on solid support media such as tissue culture plates. A large animal study or a human clinical trial may require 10(15) particles of vector, depending on dose. To generate this quantity of rAAV by transfection, more than 10(11) HEK293 cells may be needed, which would require about 5000 x 175 cm(2) flasks. The ability to scale up rAAV production by these methods severely restricts the commercialization and use of AAV vectors. A recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus is widely employed for large-scale production of heterologous proteins in cultured insect cells and may provide an attractive alternative. Toward this goal, we have explored the production of rAAV in invertebrate cells. Sf9 cells may be coinfected in suspension cultures with three recombinant baculoviruses (a Rep-baculovirus, a VP-baculovirus, and an AAV ITR vector genome baculovirus) and, 3 days later, rAAV is recovered. The particles produced are indistinguishable from 293 cell-produced rAAV, as determined on the basis of physical properties and biologic activities. Particles produced by either method were composed of similar proteins and nucleic acid. The yield of genome-containing particles produced per Sf9 cell approached 5 x 10(4), thus, 1000 ml of cultured Sf9 cells produced the equivalent of between 500 to 1000 x 175 cm(2) flasks of 293 cells. This robust system provides a simple, cost-effective method for AAV vector production.

Triple Transduction with Adeno-Associated Virus Vectors Expressing Tyrosine Hydroxylase, Aromatic-L-Amino-Acid Decarboxylase, and GTP Cyclohydrolase I for Gene Therapy of Parkinson's Disease

Parkinsons disease (PD), a neurological disease suited to gene therapy, is biochemically characterized by a severe decrease in the dopamine content of the striatum. One current strategy for gene therapy of PD involves local production of dopamine in the striatum achieved by inducing the expression of enzymes involved in the biosynthetic pathway for dopamine. We previously showed that the coexpression of tyrosine hydroxylase (TH) and aromatic-L-amino-acid decarboxylase (AADC), using two separate adeno-associated virus (AAV) vectors, resulted in more effective dopamine production and more remarkable behavioral recovery in 6-hydroxydopamine-lesioned parkinsonian rats, compared with the expression of TH alone. Not only levels of TH and AADC but also levels of tetrahydrobiopterin (BH4), a cofactor of TH, and GTP cyclohydrolase I (GCH), a rate-limiting enzymes for BH4 biosynthesis, are reduced in parkinsonian striatum. In the present study, we investigated whether transduction with separate AAV vectors expressing TH, AADC, and GCH was effective for gene therapy of PD. In vitro experiments showed that triple transduction with AAV-TH, AAV-AADC, and AAV-GCH resulted in greater dopamine production than double transduction with AAV-TH and AAV-AADC in 293 cells. Furthermore, triple transduction enhanced BH4 and dopamine production in denervated striatum of parkinsonian rats and improved the rotational behavior of the rats more efficiently than did double transduction. Behavioral recovery persisted for at least 12 months after stereotaxic intrastriatal injection. These results suggest that GCH, in addition to TH and AADC, is important for effective gene therapy of PD.

A Phase I Study of Aromatic l-Amino Acid Decarboxylase Gene Therapy for Parkinson's Disease

Gene transfer of dopamine-synthesizing enzymes into the striatal neurons has led to behavioral recovery in animal models of Parkinsons disease (PD). We evaluated the safety, tolerability, and potential efficacy of adeno-associated virus (AAV) vector-mediated gene delivery of aromatic L-amino acid decarboxylase (AADC) into the putamen of PD patients. Six PD patients were evaluated at baseline and at 6 months, using multiple measures, including the Unified Parkinsons Disease Rating Scale (UPDRS), motor state diaries, and positron emission tomography (PET) with 6-[(18)F]fluoro-L-m-tyrosine (FMT), a tracer for AADC. The short-duration response to levodopa was measured in three patients. The procedure was well tolerated. Six months after surgery, motor functions in the OFF-medication state improved an average of 46% based on the UPDRS scores, without apparent changes in the short-duration response to levodopa. PET revealed a 56% increase in FMT activity, which persisted up to 96 weeks. Our findings provide class IV evidence regarding the safety and efficacy of AADC gene therapy and warrant further evaluation in a randomized, controlled, phase 2 setting.Gene transfer of dopamine-synthesizing enzymes into the striatal neurons has led to behavioral recovery in animal models of Parkinsons disease (PD). We evaluated the safety, tolerability, and potential efficacy of adeno-associated virus (AAV) vector-mediated gene delivery of aromatic L-amino acid decarboxylase (AADC) into the putamen of PD patients. Six PD patients were evaluated at baseline and at 6 months, using multiple measures, including the Unified Parkinsons Disease Rating Scale (UPDRS), motor state diaries, and positron emission tomography (PET) with 6-[18F]fluoro-L-m-tyrosine (FMT), a tracer for AADC. The short-duration response to levodopa was measured in three patients. The procedure was well tolerated. Six months after surgery, motor functions in the OFF-medication state improved an average of 46% based on the UPDRS scores, without apparent changes in the short-duration response to levodopa. PET revealed a 56% increase in FMT activity, which persisted up to 96 weeks. Our findings provide class IV evidence regarding the safety and efficacy of AADC gene therapy and warrant further evaluation in a randomized, controlled, phase 2 setting.

Cell and gene therapy using mesenchymal stem cells (MSCs).

Mesenchymal stem cells (MSCs) are considered to be a promising platform for cell and gene therapy for a variety of diseases. First, in the field of hematopoietic stem cell transplantation, there are two applications of MSCs: 1) the improvement of stem cell engrafting and the acceleration of hematopoietic reconstitution based on the hematopoiesis-supporting ability; and 2) the treatment of severe graft-versus-host disease (GVHD) based on the immunomodulatory ability. Regarding the immunosuppressive ability, we found that nitric oxide (NO) is involved in the MSC-mediated suppression of T cell proliferation. Second, tumor-bearing nude mice were injected with luciferase-expressing MSCs. An in vivo imaging analysis showed the significant accumulation of the MSCs at the site of tumors. The findings suggest that MSCs can be utilized to target metastatic tumors and to deliver anti-cancer molecules locally. As the third application, MSCs may be utilized as a cellular vehicle for protein-supplement gene therapy. When long-term transgene expression 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 adeno-associated virus (AAV) would be particularly valuable. There will be wide-ranging applications of MSCs to frontier medical treatments in the near future.

Retroviral vector‐producing mesenchymal stem cells for targeted suicide cancer gene therapy

Mesenchymal stem cells (MSCs) are a promising vehicle for targeted cancer gene therapy because of their potential of tumor tropism. For efficient therapeutic application, we developed retroviral vector‐producing MSCs that enhance tumor transduction via progeny vector production.

A novel recombinant adeno-associated virus vaccine induces a long-term humoral immune response to human immunodeficiency virus

Recombinant adeno-associated virus (AAV) has attracted tremendous interest as a promising vector for gene delivery. In this study we have developed an HIV-1 vaccine, using an AAV vector expressing HIV-1 env, tat, and rev genes (AAV-HIV vector). A single injection of the AAV-HIV vector induced strong production of HIV-1-specific serum IgG and fecal secretory IgA antibodies as well as MHC class I-restricted CTL activity in BALB/c mice. The titer of HIV-1-specific serum IgG remained stable for 10 months. When AAV-HIV vector was coadministered with AAV-IL2 vector, the HIV-specific cell-mediated immunity (CMI) was significantly enhanced. Boosting with AAV-HIV vector strongly enhanced the humoral response. Furthermore, the mouse antisera neutralized an HIV-1 homologous strain, and BALB/c mice immunized via the intranasal route with an AAV vector expressing the influenza virus hemagglutinin (HA) gene showed protective immunity against homologous influenza virus challenge. These results demonstrate that AAV-HIV vector immunization may provide a novel and promising HIV vaccination strategy.

Rescue of hereditary form of dilated cardiomyopathy by rAAV-mediated somatic gene therapy: amelioration of morphological findings, sarcolemmal permeability, cardiac performances, and the prognosis of TO-2 hamsters.

The hereditary form comprises ≈1/5 of patients with dilated cardiomyopathy (DCM) and is a major cause of advanced heart failure. Medical and socioeconomic settings require novel treatments other than cardiac transplantation. TO-2 strain hamsters with congenital DCM show similar clinical and genetic backgrounds to human cases that have defects in the δ-sarcoglycan (δ-SG) gene. To examine the long-term in vivo supplement of normal δ-SG gene driven by cytomegalovirus promoter, we analyzed the pathophysiologic effects of the transgene expression in TO-2 hearts by using recombinant adeno-associated virus vector. The transgene preserved sarcolemmal permeability detected in situ by mutual exclusivity between cardiomyocytes taking up intravenously administered Evans blue dye and expressing the δ-SG transgene throughout life. The persistent amelioration of sarcolemmal integrity improved wall thickness and the calcification score postmortem. Furthermore, in vivo myocardial contractility and hemodynamics, measured by echocardiography and cardiac catheterization, respectively, were normalized, especially in the diastolic performance. Most importantly, the survival period of the TO-2 hamsters was prolonged after the δ-SG gene transduction, and the animals remained active, exceeding the life expectancy of animals without transduction of the responsible gene. These results provide the first evidence that somatic gene therapy is promising for human DCM treatment, if the rAAV vector can be justified for clinical use.

Adeno-associated virus vector-mediated bcl-2 gene transfer into post-ischemic gerbil brain in vivo: prospects for gene therapy of ischemia-induced neuronal death.

The proto-oncogene bcl-2 is known as an anti-apoptotic gene that confers the ability to block neuronal cell death after transient ischemia. In order to examine whether the bcl-2 gene can be used for protection of ischemic brain injury, we generated adeno-associated virus (AAV) vectors capable of expressing human bcl-2. Replication-defective AAV vectors were found effectively to transfer and express bcl-2 gene in the gerbil hippocampal neurons. Transduction with AAV bcl-2 5 days before forebrain ischemia prevented the DNA fragmentation in the CA1 neurons that is commonly associated with ischemia-induced cell death. Furthermore, the application of AAV bcl-2 as late as 1 h following an ischemic insult also prevented DNA fragmentation in CA1 neurons. These results suggest that the bcl-2 protein has neuroprotective functions that inhibit ischemic cell death and demonstrate the potential of AAV bcl-2 for use in post-ischemic gene therapy in the brain.

Interleukin-10 Expression Mediated by an Adeno-Associated Virus Vector Prevents Monocrotaline-Induced Pulmonary Arterial Hypertension in Rats

Pulmonary arterial hypertension (PAH) is a fatal disease associated with inflammation and pathological remodeling of the pulmonary artery (PA). Interleukin (IL)-10 is a pleiotropic antiinflammatory cytokine with vasculoprotective properties. Here, we report the preventive effects of IL-10 on monocrotaline-induced PAH. Three-week-old Wistar rats were intramuscularly injected with an adeno-associated virus serotype 1 vector expressing IL-10, followed by monocrotaline injection at 7 weeks old. IL-10 transduction significantly improved survival rates of the PAH rats 8 weeks after monocrotaline administration compared with control gene transduction (75% versus 0%, P<0.01). IL-10 also significantly reduced mean PA pressure (22.8±1.5 versus 29.7±2.8 mm Hg, P<0.05), a weight ratio of right ventricle to left ventricle plus septum (0.35±0.04 versus 0.42±0.05, P<0.05), and percent medial thickness of the PA (12.9±0.3% versus 21.4±0.4%, P<0.01) compared with controls. IL-10 significantly reduced macrophage infiltration and vascular cell proliferation in the remodeled PA in vivo. It also significantly decreased the lung levels of transforming growth factor-β1 and IL-6, which are indicative of PA remodeling. In addition, IL-10 increased the lung level of heme oxygenase-1, which strongly prevents PA remodeling. In vitro analysis revealed that IL-10 significantly inhibited excessive proliferation of cultured human PA smooth muscle cells treated with transforming growth factor-β1 or the heme oxygenase inhibitor tin protoporphyrin IX. Thus, IL-10 prevented the development of monocrotaline-induced PAH, and these results provide new insights into the molecular mechanisms of human PAH.

Scalable Generation of High-Titer Recombinant Adeno-Associated Virus Type 5 in Insect Cells

ABSTRACT We established a method for production of recombinant adeno-associated virus type 5 (rAAV5) in insect cells by use of baculovirus expression vectors. One baculovirus harbors a transgene between the inverted terminal repeat sequences of type 5, and the second expresses Rep78 and Rep52. Interestingly, the replacement of type 5 Rep52 with type 1 Rep52 generated four times more rAAV5 particles. We replaced the N-terminal portion of type 5 VP1 with the equivalent portion of type 2 to generate infectious AAV5 particles. The rAAV5 with the modified VP1 required α2-3 sialic acid for transduction, as revealed by a competition experiment with an analog of α2-3 sialic acid. rAAV5-GFP/Neo with a 4.4-kb vector genome produced in HEK293 cells or Sf9 cells transduced COS cells with similar efficiencies. Surprisingly, Sf9-produced humanized Renilla green fluorescent protein (hGFP) vector with a 2.4-kb vector genome induced stronger GFP expression than the 293-produced one. Transduction of murine skeletal muscles with Sf9-generated rAAV5 with a 3.4-kb vector genome carrying a human secreted alkaline phosphatase (SEAP) expression cassette induced levels of SEAP more than 30 times higher than those for 293-produced vector 1 week after injection. Analysis of virion DNA revealed that in addition to a 2.4- or 3.4-kb single-stranded vector genome, Sf9-rAAV5 had more-abundant forms of approximately 4.7 kb, which appeared to correspond to the monomer duplex form of hGFP vector or truncated monomer duplex SEAP vector DNA. These results indicated that rAAV5 can be generated in insect cells, although the difference in incorporated virion DNA may induce different expression patterns of the transgene.