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. 2005 Jan;79(1):214-24.
doi: 10.1128/JVI.79.1.214-224.2005.

Unrestricted hepatocyte transduction with adeno-associated virus serotype 8 vectors in mice

Hiroyuki Nakai  1 , Sally Fuess, Theresa A Storm, Shin-ichi Muramatsu, Yuko Nara, Mark A Kay
Affiliations

Unrestricted hepatocyte transduction with adeno-associated virus serotype 8 vectors in mice

Hiroyuki Nakai et al. J Virol. 2005 Jan.

Abstract

Recombinant adeno-associated virus (rAAV) vectors can mediate long-term stable transduction in various target tissues. However, with rAAV serotype 2 (rAAV2) vectors, liver transduction is confined to only a small portion of hepatocytes even after administration of extremely high vector doses. In order to investigate whether rAAV vectors of other serotypes exhibit similar restricted liver transduction, we performed a dose-response study by injecting mice with beta-galactosidase-expressing rAAV1 and rAAV8 vectors via the portal vein. The rAAV1 vector showed a blunted dose-response similar to that of rAAV2 at high doses, while the rAAV8 vector dose-response remained unchanged at any dose and ultimately could transduce all the hepatocytes at a dose of 7.2 x 10(12) vector genomes/mouse without toxicity. This indicates that all hepatocytes have the ability to process incoming single-stranded vector genomes into duplex DNA. A single tail vein injection of the rAAV8 vector was as efficient as portal vein injection at any dose. In addition, intravascular administration of the rAAV8 vector at a high dose transduced all the skeletal muscles throughout the body, including the diaphragm, the entire cardiac muscle, and substantial numbers of cells in the pancreas, smooth muscles, and brain. Thus, rAAV8 is a robust vector for gene transfer to the liver and provides a promising research tool for delivering genes to various target organs. In addition, the rAAV8 vector may offer a potential therapeutic agent for various diseases affecting nonhepatic tissues, but great caution is required for vector spillover and tight control of tissue-specific gene expression.

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Figures

FIG. 1.
FIG. 1.
Vector dose-response profiles in AAV1-, AAV2-, and AAV8-EF1α-nlslacZ-transduced mouse livers. The percentage of transduced hepatocytes in the livers (A), total β-galactosidase antigen levels (B), and number of double-stranded vector genomes per diploid genomic equivalent (ds-vg/dge) (C) are shown as a function of injected vector doses. Solid markers represent the values obtained from the present study. The dose-response profiles in AAV2-EF1α-nlslacZ-mediated liver transduction were obtained from our previous study (39) for comparison and are depicted with open circles. Values are means ± standard deviation.
FIG. 2.
FIG. 2.
Liver transduction with 7.2 × 1012 vg of AAV8-EF1α-nlslacZ delivered via the portal vein. The liver was harvested 6 weeks postinjection and stained with X-Gal and light hematoxylin. A representative result is shown (A and B). Virtually all hepatocytes were transduced with rAAV8 throughout the liver. The liver was stained heterogeneously with X-Gal, with central vein areas being less intense. (C) X-Gal-stained hepatocytes around a central vein area. Although gene expression near central veins was not as strong as in portal areas, most of the hepatocytes express the transgene. (D) X-Gal and F4/80 double staining. None of the Kupffer cells (brown) were transduced. Small nuclei positive for β-galactosidase are indicated with arrows in panels B and D. These might be portions of hepatocyte nuclei or represent rAAV8-transduced nonparenchymal cells besides Kupffer cells. Scale bars, 100 μm.
FIG. 3.
FIG. 3.
Southern blot analysis of rAAV vector genomes in liver transduced with AAV1- or AAV8-EF1α-nlslacZ at various doses. The left and right panels show the results obtained with AAV1-EF1α-nlslacZ- and AAV8-EF1α-nlslacZ-injected mice. Total genomic DNA was extracted from the livers harvested 6 weeks postinjection and separated on 0.8% agarose gels following BamHI or KpnI digestion. BamHI cleaves the vector genome only once at nucleotide position 1362, while KpnI does not cut the 4,828-base genome. The vector genomes were detected with a 2.1-kb lacZ probe (nucleotide positions 1518 to 3639). Each lane represents an individual mouse. Injected vector doses (vg per mouse) are indicated above each lane. For the results obtained from the mice injected with 5.0 × 1010 vg/mouse, strips from overexposed blots are also shown to demonstrate the presence or absence of concatemers. They are indicated with thicker lines above the lanes. Open and solid arrows indicate head-to-tail and tail-to-tail molecules, respectively. Open and solid arrowheads indicate supercoiled double-stranded circular monomer vector genomes and concatemers, respectively. Head-to-tail molecules include both circular monomer genomes and concatemers, while tail-to-tail molecules represent concatemers exclusively. Therefore, the intensity of tail-to-tail molecules well correlates with the abundance of concatemers.
FIG. 4.
FIG. 4.
Comparison of efficiency of rAAV8-mediated liver transduction between tail vein and portal vein injections. (A) Plasma human coagulation factor IX (hF.IX) levels after tail vein (TV) or portal vein (PV) injection of AAV8-hF.IX16 into male C57BL/6 mice. Robust human coagulation factor IX expression with no lag phase was observed with both routes. Expression peaked 4 weeks after injection, followed by a substantial (≈75%) decline. Vertical bars indicate standard deviations. (B) Vector genome copy numbers (ds-vg/dge) in livers transduced with AAV8-EF1α-nlslacZ via tail vein or portal vein injection at 3.0 × 1011 or 7.2 × 1012 vg/mouse. Total liver DNA was extracted 6 weeks postinjection, and 10 μg of DNA was analyzed by Southern blot with BglI digestion and a 2.1-kb lacZ probe (BglI-BglI fragment). The left and right blots were analyzed separately with a different series of vector copy number standards. The double-stranded vector copy number standards (0 to 100 and 0 to 1,000 ds-vg/dge) were prepared by adding the corresponding amount of plasmid, pAAV-EF1α-nlslacZ, to 10 μg of liver DNA extracted from a naïve mouse. Each lane represents an individual mouse. Routes of administration and vector doses are indicated above the lanes.
FIG. 5.
FIG. 5.
Representative photomicrographs of sections of various mouse tissues 6 weeks after tail vein injection of AAV8-EF1α-nlslacZ at a dose of 7.2 × 1012 vg/mouse (A to D) or 3 weeks after tail vein injection of AAV8-CMV-lacZ at a dose of 3.0 ×1011 or 7.2 × 1012 vg/mouse (E). The sections were either X-Gal stained (A, B, D, and E) or stained with designated antibodies (C). (A) Tissue distribution of β-galactosidase-positive cells: lu, lung; h, heart; s, spleen; k, kidney; i, intestine; t, testis; p, pancreas; and m, skeletal muscle (quadriceps). The top row represents tissues from a mouse injected with excipient only, while the bottom row shows samples from vector-injected mice. (B) Brain transduction with rAAV8. (a) Cerebral cortex. Positive cells are scattered throughout the region. (b) Hippocampus. Positive cells are observed in both granule and pyramidal cell layers. (c) Striatum. (d) Amygdala. (e) Hypothalamus. β-Galactosidase-positive neurons and glial cells are clustered in the arcuate nucleus and median eminence. Some ependymal cells of the third ventricle are also positive. (f) Cerebellum. Purkinje cells are regionally well transduced. (C) Confocal microscopy to assess colocalization of β-galactosidase and either NeuN (a marker for neurons) or GFAP (a marker for astrocytes) to determine rAAV8-transduced cell types in the cerebral cortex of the brain. Both neurons and glial cells were transduced with rAAV8. Scale bars, 5 μm. (D) Transduction of vascular smooth muscle cells in the walls of a branch of the coronary artery (a) and a branch of the splenic artery (b). (E) Tissue distribution of β-galactosidase-positive cells in mice injected with AAV8-CMV-lacZ via the tail vein. The vector doses (vg/mouse) are indicated above the pictures. The section of the pancreas was also stained with anti-insulin antibody (brown cells). Scale bars (duplicated lines), 250 μm. lv, liver; t.a., tibialis anterior limb muscle. The tissues in panels A, B, D, and E were counterstained with nuclear fast red or light hematoxylin. Scale bars represent 100 μm unless otherwise noted.
FIG. 6.
FIG. 6.
Tissue distribution analysis by Southern blot. Various tissues were harvested from mice injected with 7.2 × 1012 vg of AAV8-EF1α-nlslacZ via the tail vein or the portal vein (one mouse each). Total genomic DNA was extracted from tissues, and 10 μg of each DNA was digested with BglI and separated on a 0.8% agarose gel. The vector genomes were detected with a 2.1-kb BglI-BglI lacZ probe. The double-stranded vector genome copy number standards (0 to 30 ds-vg/dge) were prepared as described in the legend to Fig. 4. Abbreviations: b, brain; lu, lung; h, heart; s, spleen; k, kidney; i, intestine; t, testis; p, pancreas; and m, skeletal muscle. In each set of tissues, the left and right lanes represent samples from mice injected via the tail vein and portal vein, respectively. For densitometric analysis, see the Results section.
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