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

Urabe, Masashi; Nakakura, Takayo; Xin, Ke-Qin; Obara, Yoko; Mizukami, Hiroaki; Kume, Akihiro; Kotin, Robert M.; Ozawa, Keiya

doi:10.1128/jvi.80.4.1874-1885.2006

Cited by 95 publications

(101 citation statements)

References 39 publications

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“…Furthermore, Takeuchi et al have recently discussed the VP1-VP2 boundary as a recombination hot spot (29). Two recent attempts to produce AAV vectors in insect cells have shown that swapping the AAV2 VP1 region with the corresponding regions of AAV5 and AAV8 can alter the viral tropism (30,31). These studies along with our data highlight modularity of this critical capsid domain.…”

Section: Discussionsupporting

confidence: 55%

Directed evolution of adeno-associated virus to an infectious respiratory virus

Excoffon

Koerber²,

Dickey

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

155

180

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Respiratory viruses evolve to maintain infectivity levels that permit spread yet prevent host and virus extinction, resulting in surprisingly low infection rates. Respiratory viruses harnessed as gene therapy vectors have illustrated this limitation. We used directed evolution in an organotypic human airway model to generate a highly infectious adeno-associated virus. This virus mediated gene transfer more than 100-fold better than parental strains and corrected the cystic fibrosis epithelial Cl ؊ transport defect. Thus, under appropriate selective pressures, viruses can evolve to be more infectious than observed in nature, a finding that holds significant implications for designing vectors for gene therapy and for understanding emerging pathogens.

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Section: Discussionsupporting

confidence: 55%

Directed evolution of adeno-associated virus to an infectious respiratory virus

Excoffon

Koerber²,

Dickey

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

155

180

View full text Add to dashboard Cite

show abstract

“…For example, solving the Poisson distribution for a particular combination of individual BEV helpers (e.g., 3:3:3) predicts the ratio of the respectively infected cells to be only 1.1%. The specifics of rAAV production also requires a coordinated and timely expression of 7 helper Rep and Cap proteins at the optimal stoichiometry (2,5,16,17). Adding to the complexity of the system is the fact of the apparent instability of the recombinant BEVs, especially the Bac-Rep helper (5).…”

Section: Discussionmentioning

confidence: 99%

An inducible system for highly efficient production of recombinant adeno-associated virus (rAAV) vectors in insect Sf9 cells

Aslanidi

Lamb

Zolotukhin

2009

Proc. Natl. Acad. Sci. U.S.A.

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Production of clinical-grade gene therapy vectors for human trials remains a major hurdle in advancing cures for a number of otherwise incurable diseases. We describe a system based on a stably transformed insect cell lines harboring helper genes required for vector production. Integrated genes remain silent until the cell is infected with a single baculovirus expression vector (BEV). The induction of expression results from a combination of the amplification of integrated resident genes (up to 1,200 copies per cell) and the enhancement of the expression mediated by the immediate-early trans-regulator 1 (IE-1) encoded by BEV. The integration cassette incorporates an IE-1 binding target sequence from wild-type Autographa californica multiple nuclear polyhedrosis virus, a homologous region 2 (hr2). A feed-forward loop is initiated by one of the induced proteins, Rep78, boosting the amplification of the integrated genes. The system was tested for the coordinated expression of 7 proteins required to package recombinant adeno-associated virus (rAAV)2 and rAAV1. The described arrangement provided high levels of Rep and Cap proteins, thus improving rAAV yield by 10-fold as compared with the previously described baculovirus/rAAV production system. baculovirus ͉ gene therapy

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“…On verification, the pAAV-scFvA␤ KDEL IB and pAAV-scFvphe KDEL IB plasmids were packaged into serotype-2 virions using a previously described baculovirus-based methodology. 20 …”

Section: Construction Verification and Packaging Of Ib-expressing Rmentioning

confidence: 99%

“…20 To confirm the AD pathology status at the ages examined in the current study, immunohistochemistry was performed on the brains of 3ϫTg-AD and Non-Tg mice for amyloid precursor protein (APP), intracellular and extracellular A␤ , human tau, and phosphorylated human tau protein. Qualitative analysis of marker expression revealed no alterations in the expression of human APP in the CA1 region of the brains of 2-and 6-month-old 3ϫTg-AD mice ( Figure 2, A and B).…”

Section: Ad-related Pathology In the Brains Of 3ϫtg-ad Micementioning

confidence: 99%

Early Oligodendrocyte/Myelin Pathology in Alzheimer's Disease Mice Constitutes a Novel Therapeutic Target

Desai

Mastrangelo

Ryan

et al. 2010

The American Journal of Pathology

182

173

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The detection of myelin disruptions in Alzheimer's disease (AD)-affected brain raises the possibility that oligodendrocytes undergo pathophysiological assault over the protracted course of this neurodegenerative disease. Oligodendrocyte compromise arising from direct toxic effects imparted by pathological amyloid-␤ peptides and/or through signals derived from degenerating neurons could play an important role in the disease process. We previously demonstrated that 3؋Tg-AD mice, which harbor the human amyloid precursor protein Swedish mutant transgene, presenilin knock-in mutation, and tau P301L mutant transgene, exhibit significant alterations in overall myelination patterns and oligodendrocyte status at time points preceding the appearance of amyloid and tau pathology. Herein, we demonstrate that A␤ 1-42 leads to increased caspase-3 expression and apoptotic cell death of both nondifferentiated and differentiated mouse oligodendrocyte precursor (mOP) cells in vitro. Through use of a recombinant adeno-associated virus serotype-2 (rAAV2) vector expressing an A␤ 1-42 -specific intracellular antibody (intrabody), oligodendrocyte and myelin marker expression, as well as myelin integrity, were restored in the vector-infused brain regions of 3؋Tg-AD mice. Overall, this work provides further insights into the impact of A␤ 1-42 -mediated toxicity on the temporal and spatial progression of subtle myelin disruption during the early presymptomatic stages of AD and may help to validate new therapeutic options designed to avert these early impairments.

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

Cited by 95 publications

References 39 publications

Directed evolution of adeno-associated virus to an infectious respiratory virus

Directed evolution of adeno-associated virus to an infectious respiratory virus

An inducible system for highly efficient production of recombinant adeno-associated virus (rAAV) vectors in insect Sf9 cells

Early Oligodendrocyte/Myelin Pathology in Alzheimer's Disease Mice Constitutes a Novel Therapeutic Target

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