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Review
. 2003:55:3-64.
doi: 10.1016/s0074-7742(03)01001-8.

Nonneurotropic adenovirus: a vector for gene transfer to the brain and gene therapy of neurological disorders

Pedro R Lowenstein  1 , Donata Suwelack, Jinwei Hu, Xianpeng Yuan, Maximiliano Jimenez-Dalmaroni, Shyam Goverdhana, Maria G Castro
Affiliations
Review

Nonneurotropic adenovirus: a vector for gene transfer to the brain and gene therapy of neurological disorders

Pedro R Lowenstein et al. Int Rev Neurobiol. 2003.
No abstract available

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Figures

Fig. 1
Fig. 1
(A) Schematic representation of an adenovirus particle based on the current understanding of its polypeptide constituents and genome. (B) Genome is 100 map units (mu) in length (1 mu = 360 bp). Early primary mRNA transcripts are designated in bold. Certain polypeptides are identified by conventional numbering system (roman numerals). Modified from Flint et al. (2000). (See Color Insert.)
Fig. 2
Fig. 2
An E1-, E3-deleted helper adenovirus (FL helper) with a packaging signal sensitive to FLP3-mediated excision. (A) FL helper was generated by homologous recombination in 293 cells after cotransfection with plasmid pΔE1sp1A-2xFRT and pBHG10-CMVluc-I. The bottom half of the figure indicates the left-end region of the helper virus genome, before and after FLP3-mediated recombination; the figure also indicates the location of the PCR primers used to determine excision of the packaging signal, ψ (flanked by black arrows). (B) Production of gutless adenoviral vectors. Diagram of an ideal gene delivery vector for neurological gene therapy using the “gutless” adenoviral vector. The packaging signal of the helper virus, flanked by FRT sites, is excised after growth in 293 cells expressing FLPe recombinase. The cloning capacity of this system (N30 kbp) will enable the simultaneous use of two or more therapeutic genes and also includes inducible promoter systems. (See Color Insert.)
Fig. 3
Fig. 3
Transgene expression (β-galactosidase) and inflammation in the brains of animals injected with either the E1/E3-deleted vector RAd35 or the HC-Adv AdGS46. Rows a and b show β-galactosidase expression (columns 1 and 2), CD8+ cell infiltration (column 3), MHC class I upregulation (column 4), and microglial cell activation (ED1, column 5), 3 days after injection of 1 × 107 IU of RAd35 (row a) or AdGS46 (row b) into the brains of naive animals. Levels of transgene expression and inflammation mediated by the E1/E3-deleted, or the HC-Adv vector, were indistinguishable in naive animals. Transgene expression from 1 × 107 IU of both RAd35 and AdGS46 is stable for at least 30 days in naive animals and inflammation resolves within this time frame (shown in row c only for AdGS46). However, transgene expression from E1/E3-deleted vectors in the CNS is rapidly eliminated and accompanied by severe brain inflammation if animals receive a subsequent peripheral infection with adenovirus [row d; RAd35 injected in the brain at Day 0, RAdHPRT (Southgate et al., 1999) injected in the skin at Day 60. See also the quanitative analysis of all markers, in panel f]. In contrast, transgene expression from HC-Adv remains stable, and no brain inflammation is elicited when animals are subsequently injected with RAdHPRT in the skin (row e; AdGS46 injected in the brain at Day 0, RAdHPRT injected in the skin at Day 60).
Fig. 4
Fig. 4
Preimmunization against adenovirus eliminates rapidly transgene expression from E1/E3-deleted vectors, but not from high-capacity vectors. Quantification of the area within 40-μm-thick brain sections occupied by β-galactosidase and ED1. Error bars show the SEM value from the five animals in each experimental group. Student’s t test was used to calculate the degree of significance of differences between levels of transgene expression and inflammation in the brains of nonimmunized animals (black bars) and immunized animals (hatched bars) after intrastriatal injection of RAd35 (right) or AdGS46 (left). The x-axis indicates the days after vector injection; vector was injected on day 0, and animals were immunized 14 days before the brain injection.
Fig. 5
Fig. 5
In vivo β-galactosidase expression in rat brain infected with either GS46-FLP HC-Adv vector or E1/E3-deleted adenovirus (RAd35). 7 × 107 BFU of either virus (in 3 μl) was injected into the striatum of male Sprague–Dawley rats. Six days after virus inoculation, β-galactosidase expression was analyzed by immunohistochemistry. (a, b) GS46-FLP HD virus. (c, d) RAd35 virus. β-Galactosidase is expressed under the control of the hCMV promoter in both viruses. From Umana et al. (2001b).
Fig. 6
Fig. 6
The mCMV promoter allows high-level expression at low doses of vector, without inflammation. Increasing doses of RAd36 (mCMV-βgal) (104–106) were injected into the striatum of adult Sprague–Dawley rats in a total volume of 3 μl, and animals were perfused 5 days later. Brains were processed for immunohistochemistry to detect β-galactosidase (transgene expression; first two left hand side columns), ED1 (macrophages-microglial cells), and CD8 (infiltrating lymphocytes and NK cells). Low-power view of the striatum is shown in the left-hand side column, and a higher power view is shown in the column next to it. Scale bar shown in the upper left = 1 mm; all others = 100 μm. RAd36 allows strong transduction of the striatum, in the absence of recruitment of inflammatory cells, compared to animals injected with saline controls. This image illustrates three doses of animals injected with RAd36. The quantification of this experiment is shown in Fig. 4, above.
See this image and copyright information in PMC

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