1. Global non-viral gene transfer to the primate brain following intravenous administration 
Yun Zhang, Felix Schlachetzki, William M Pardridge 
Molecular Therapy 
Volume 7, Issue 1, Pages (January 2003)
DOI: /S (02)
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
The plasmid DNA is encapsulated in the interior of an 85 nm pegylated immunoliposome (PIL). The surface of the liposome is conjugated with several thousand strands of 2000 Dalton polyethyleneglycol (PEG), and the tips of 1–2% of the PEG strands is tethered with a targeting ligand, such as a insulin receptor (IR)-specific monoclonal antibody (MAb). The MAb-conjugated PIL undergoes receptor-mediated transcytosis across the blood-brain barrier and receptor-mediated endocytosis across the brain cell plasma membrane and enters the nuclear compartment [12].
Molecular Therapy 2003 7, 11-18DOI: ( /S (02) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Luciferase gene expression in the brain and other organs of the adult rhesus monkey (A) and adult rat (B) measured at 48 hours after a single intravenous injection of the PIL carrying the plasmid DNA. Data are mean ± SE. The plasmid DNA encoding the luciferase gene used in either species is clone 790, which is driven by the SV40 promoter [11,12]. The PIL carrying the DNA was targeted to primate organs with the HIRMAb and to rat organs with the OX26 TfRMAb.
Molecular Therapy 2003 7, 11-18DOI: ( /S (02) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
β-Galactosidase histochemistry of brain removed from either the HIRMAb-PIL injected rhesus monkey (A, C, D, E, and F) or the control, uninjected rhesus monkey (B). The plasmid DNA encapsulated in the PIL is the pSV-β-galactosidase expression plasmid driven by the SV40 promoter. Panel A is a reconstruction of the 2 halves of a coronal section of the forebrain. Panel C shows half-coronal sections through the primate cerebrum and a full coronal section through the cerebellum; the sections from top to bottom are taken from the rostral to caudal parts of brain. Panels D, E, and F are light micrographs of choroid plexus, occipital cortex, and cerebellum, respectively. All specimens are β-galactosidase histochemistry without counter-staining. The magnification in panels A and B is the same and the magnification bar in panel A is 3 mm; the magnification bar in panel C is 8 mm; the magnification bars in panels D–F are 155 μm.
Molecular Therapy 2003 7, 11-18DOI: ( /S (02) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Confocal immunofluorescent imaging of β-galactosidase expression in neurons of the gene injected (A–F) and the uninjected control rhesus monkey brain (G–I). Immunoreactive neuronal nuclei (NeuN) are stained with a mouse anti-neuN primary antibody and a fluorescein-labeled secondary antibody, as shown in panels A, D, and G. Immunoreactive β-galactosidase is stained with a rabbit anti-bacterial β-galactosidase primary antibody and a rhodamine-labeled secondary antibody, as shown in panels B, E, and H. The respective superimposed yellow images reveal co-localization of the neuN and β-galactosidase in neurons (C, F). Primate brain was also immunolabeled with the isotype control mouse IgG1 or rabbit IgG, which showed no specific staining as shown in the insets of panels A and B, respectively. Scale bars in panels A–C and G–I are 100 μm; scale bars in panels D–F (oil immersion) are 50 μm. Neurons from the primate supplementary motor cortex are shown in panels A–C, and larger neurons from the primate primary motor cortex are shown in panels G–H.
Molecular Therapy 2003 7, 11-18DOI: ( /S (02) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
β-Galactosidase histochemistry of rhesus monkey organs removed 48 hours after intravenous injection of the pSV-β-galactosidase expression plasmid encapsulated in the HIRMAb-PIL, including liver (A,B), spleen (C, D), heart (E, F), biceps skeletal muscle (G, H), and omental fat (I, J). Magnification in panels A, C, E, G, and I is the same and the magnification bar in panel A is 5 mm. Magnification in panels B, D, F, H, and J is the same and the magnification bar in panel B is 65 μm. The β-galactosidase gene is diffusely expressed in primate liver and spleen, whereas there is no detectable gene expression in heart, skeletal muscle, or fat. No sections are counter-stained.
Molecular Therapy 2003 7, 11-18DOI: ( /S (02) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions