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. 2000 Jun 20;97(13):7567-72.
doi: 10.1073/pnas.130187497.

Noninvasive Gene Targeting to the Brain

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Free PMC article

Noninvasive Gene Targeting to the Brain

N Shi et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Gene therapy of the brain is hindered by the presence of the blood-brain barrier (BBB), which prevents the brain uptake of bloodborne gene formulations. Exogenous genes have been expressed in the brain after invasive routes of administration, such as craniotomy or intracarotid arterial infusion of noxious agents causing BBB disruption. The present studies describe the expression of an exogenous gene in brain after noninvasive i.v. administration of a 6- to 7-kb expression plasmid encoding either luciferase or beta-galactosidase packaged in the interior of neutral pegylated immunoliposomes. The latter are conjugated with the OX26 mAb to the rat transferrin receptor, which enables targeting of the plasmid DNA to the brain via the endogenous BBB transferrin receptor. Unlike cationic liposomes, this neutral liposome formulation is stable in blood and does not result in selective entrapment in the lung. Luciferase gene expression in the brain peaks at 48 h after a single i.v. administration of 10 microg of plasmid DNA per adult rat, a dose that is 30- to 100-fold lower than that used for gene expression in rodents with cationic liposomes. beta-Galactosidase histochemistry demonstrated gene expression throughout the central nervous system, including neurons, choroid plexus epithelium, and the brain microvasculature. In conclusion, widespread gene expression in the brain can be achieved by using a formulation that does not employ viruses or cationic liposomes, but instead uses endogenous receptor-mediated transport pathways at the BBB.

Figures

Figure 1
Figure 1
(A) Scheme showing plasmid (DNA) encapsulated in pegylated immunoliposomes constructed from neutral lipids. There are approximately 3000 strands of polyethylene glycol of 2000 Da molecular mass, designated PEG 2000, attached to the liposome surface, and about 1% of the PEG strands is conjugated with an mAb to a BBB receptor. (B) The mean diameter of the pegylated liposomes encapsulating the pGL2 plasmid DNA is 73 nm. (C) Liposomes before (lane 2) and after (lane 1) DNase 1/exonuclease III treatment were resolved with 0.8% agarose gel electrophoresis followed by ethidium bromide (Et Br) staining. DNA molecular weight size standards are shown in the left hand side. Approximately 50% of the DNA associated with the pegylated liposome was bound to the exterior of the liposome (lane 2), and the exteriorized DNA was quantitatively removed by the nuclease treatment (lane 1). A trace amount of the pGL2 plasmid was radiolabeled with 32P, and film autoradiography of the gel showed a single 5.8-kb band with no low molecular weight radiolabeled DNA. (D) The conjugation of the OX26 mAb to the pegylated liposomes carrying the encapsulated pGL2 plasmid DNA after nuclease digestion is demonstrated by Sepharose CL-4B gel filtration chromatography. A trace amount of the encapsulated pGL2 plasmid DNA was labeled with 32P, and a trace amount of the OX26 mAb was radiolabeled with 3H. The study shows the comigration of the conjugated OX26 mAb and the encapsulated pGL2 plasmid DNA.
Figure 2
Figure 2
(left) The percentage of injected dose (ID) per ml plasma that is precipitated by TCA is plotted vs. the time after i.v. injection of the 32P DNA in anesthetized rats for up to 120 min. The DNA was injected 1 of 3 formulations: (i) “naked” DNA (DNA), (ii) pGL2 plasmid DNA encapsulated within the interior of nuclease-treated OX26-pegylated immunoliposomes (OX26-Lipo/DNA), and (iii) pGL2 plasmid DNA encapsulated in the interior of nuclease-treated pegylated liposomes without OX26 mAb attached (Peg-Lipo/DNA) (Right). The percentage of plasma radioactivity that is precipitated by TCA is shown. Data mean are ± SE (n = 3 rats/group).
Figure 3
Figure 3
The tissue uptake, expressed as percentage injected dose (ID) per gram of tissue, for liver, brain, kidney, or heart is shown at 120 min after i.v. injection of the encapsulated 32P pGL2 plasmid DNA incorporated in either pegylated liposomes without Ab attached (PEG/DNA) or within the OX26 pegylated immunoliposomes (OX26-Lipo/DNA). Data are mean ± SE (n = 3 rats/group).
Figure 4
Figure 4
The organ luciferase activity, expressed as relative light units (RLU) per mg of tissue protein, is shown for brain, heart, kidney, liver, lung, and spleen at 24, 48, and 72 h after injection of the pGL2 plasmid DNA encapsulated in pegylated immunoliposomes that were conjugated with the OX26 mAb. Data are mean ± SE (n = 3 rats/group).
Figure 5
Figure 5
β-galactosidase histochemistry in brain (AE) and liver (F) at 48 h after i.v. injection of the β-galactosidase gene packaged inside the OX26 pegylated immunoliposome (BF). The control brain from rats receiving no gene administration is shown in B. Magnification bars = 1.5 mm (A), 2.2 mm (B), 57 μm (C), 23 μm (D), 230 μm (E), and 15 μm (F). A and B were not counterstained. The lateral ventricles (LV), third ventricle (III), left or right hippocampus (hippo), and hypothalamic supraoptic nuclei (son) are labeled in A. C shows punctate gene expression in intraparenchymal capillaries and may represent gene expression in either endothelium or microvascular pericytes. (D) Gene expression in the epithelium of the choroid plexus. The lumen (L) of a capillary of the choroid plexus is shown and demonstrates the absence of β-galactosidase enzyme activity in the plasma compartment. (E) The thalamic (thal) nuclei below the choroid plexus of the third ventricle, which is also visible in A. (F) Abundant gene expression in hepatocytes; this high magnification shows a speckled pattern, suggesting localization of the β-galactosidase enzyme within the liver cell endoplasmic reticulum.

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