Targeted delivery of proteins acrossthe blood–brain barrierBrian J. Spencer* and Inder M. Verma†

Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA 92037

Contributed by Inder M. Verma, March 8, 2007 (sent for review February 12, 2007)

Treatment of many neuronal degenerative disorders will requiredelivery of a therapeutic protein to neurons or glial cells across thewhole CNS. The presence of the blood–brain barrier hampers thedelivery of these proteins from the blood, thus necessitating a newmethod for delivery. Receptors on the blood–brain barrier bindligands to facilitate their transport to the CNS; therefore, wehypothesized that by targeting these receptors, we may be able todeliver proteins to the CNS for therapy. Here, we report the use ofthe lentivirus vector system to deliver the lysosomal enzymeglucocerebrosidase and a secreted form of GFP to the neurons andastrocytes in the CNS. We fused the low-density lipoproteinreceptor-binding domain of the apolipoprotein B to the targetedprotein. This approach proved to be feasible for delivery of theprotein and could possibly be used as a general method for deliveryof therapeutic proteins to the CNS.

fusion protein � lentiviral vectors � low-density lipoprotein receptor

The blood–brain barrier (BBB) controls the passage of sub-stances from the blood into the CNS. Thus, a major challenge

for treatment of brain disorders is to overcome the impedimentof delivery of therapeutic macromolecules to the brain. Althoughdirect injection of therapeutic proteins may be a reasonableapproach for treatment of localized neural degenerative disor-ders that involve discrete anatomical structures within the brain(1), the treatment of many neurological disorders requires thedelivery of a therapeutic protein or peptide to the whole CNS.Delivery of these proteins is hampered by the tight regulation ofthe BBB. Current protocols for delivery of viral vector-mediatedgene delivery involve the stereotaxic injection of the vector to theCNS, resulting only in localized gene expression (2). The smallsize of the mouse may allow more widespread expression with asfew as five injections across the whole brain; however, the largersize of the human brain would require far too many injections tobe clinically feasible. Thus a new approach for targeting theseproteins to the CNS across the BBB is required for futuretreatments of widespread neural degenerative conditions.

Vascular distribution of a therapeutic protein would be apreferred method. The human brain contains on the order of 100million capillaries containing a surface area of �12 m2 (3).Nearly every neuron in the brain has its own capillary, with anaverage distance from capillary to neuron of 8–20 �m (4). Thus,delivery of a therapeutic protein to neurons across the capillarymembrane would be a method of choice. However, delivery ofproteins by vascular distribution to the CNS is not possible dueto the presence of the BBB, which is composed of a tightly sealedlayer of endothelial cells and numerous astrocytic processes thatregulate the passage and diffusion of proteins such as growthfactors from the blood stream to the CNS. Small molecules onthe order of 400–500 Da as well as some small lipid-solubleproteins can pass across the BBB unassisted; however, transportof almost all larger proteins to the CNS occurs via receptor-mediated transcytosis to the CNS (5). Well characterized BBBreceptors include the following: low-density lipoprotein (LDL)receptor (LDLR), transferrin receptor, and insulin-like growthfactor receptor (6).

The LDLR family is a group of cell-surface receptors thatbind lipoprotein complexes for internalization to the lyso-somes. The family is composed of �10 different receptors thatare expressed in a tissue-specific manner and primarily bindapolipoprotein complexes (7–9). The apolipoproteins, ofwhich the two most prominent members are apolipoprotein B(ApoB) and apolipoprotein E (ApoE), function to bind lipidsin the blood stream and target them for lysosomal degradation.Apolipoproteins bind to the LDLR on the cell surface of thetargeted cell, and then the complex is endocytosed. Conversionto an early endosome and subsequent lowering of the com-partmental pH result in release of the apolipoprotein andrecycling of the receptor to the cell surface. In contrast, at theBBB, LDLR binds apolipoproteins, resulting in transcytosis tothe abluminal side of the BBB, where, presumably, the apo-lipoprotein is released to be taken up by neurons and/orastrocytes (reviewed in refs. 3 and 10).

We therefore hypothesized that if a secreted form of aprotein that can bind to the LDLR can be produced in onecentral location or depot organ, such as muscle or liver, uponrelease in the blood stream, then the recombinant protein willbind to the LDLR and transcytose to the CNS. In this instance,the lentivirus (LV) vector system can prove useful because i.v.and i.p. delivery of a nonreplicating LV vector efficientlydelivers genes to the liver and spleen, allowing these organs tofunction as the sites of expression and secretion of a thera-peutic protein (11).

ResultsThe LDLR-binding domain of ApoB was fused to the C terminusof a mouse cDNA capable of generating the secreted form ofGFP (sGFPmApoB) or the secreted form of glucocerebrosidase(sGCmApoB; the enzyme deficient in the lysosomal disorderGaucher’s disease) (Fig. 1). This recombinant protein can beexpressed from the LV vector delivered by a single i.p. injection.To determine the extent of delivery of sGCmApoB and sGFP-mApoB to the CNS, adult mice (�8 weeks) were used to mimicthe BBB environment observed in the patient (12).

Mice were examined 14 days after vector delivery to allow forexpression and uptake of the recombinant protein, withoutallowing sufficient time for a strong antibody response to thenewly introduced protein to commence. Recombinant protein

Author contributions: B.J.S. and I.M.V. designed research; B.J.S. performed research; B.J.S.analyzed data; and B.J.S. and I.M.V. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.

Abbreviations: ApoB, apolipoprotein B; ApoE, apolipoprotein E; BBB, blood–brain barrier;GFAP, glial fibrillary acidic protein; LAMP, lysosomal-associated membrane protein; LDL,low-density lipoprotein; LDLR, LDL receptor; LV, lentivirus; sGC, secreted glucocerebrosi-dase; sGFP, secreted GFP; sGFPmApoB, sGFP myc epitope-tagged ApoB; sGCmApoB, sGCmyc epitope-tagged ApoB.

See Commentary on page 7315.

*Present address: Department of Neuroscience, University of California at San Diego, La Jolla,CA 92093.

†To whom correspondence should be addressed. E-mail: verma@salk.edu.

© 2007 by The National Academy of Sciences of the USA

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was easily visible in the liver and spleen of mice that had beeninjected with either the LV–sGCm or LV–sGCmApoB vectors(Fig. 2). In the liver, recombinant protein was observed primarilyin cells lining the hepatic sinuses, such as perisinusoidal cells,Kupffer cells, and endotheliocytes, and, to a lesser extent, inhepatocytes, as noted previously (11). In the spleen, germinalcenters appeared to contain the majority of recombinant pro-tein. Because of the small cytoplasm-to-nucleus ratio of thesecells, we have tentatively identified them as lymphocytes. Theredid not appear to be any recombinant protein taken up in thecells of the lung. Sections from animals injected with saline didnot show staining in any tissue (Fig. 2 G–I).

The recombinant sGCmApoB and sGFPmApoB, stained withmyc antibody (green), showed extensive uptake of the proteinsin the CNS only when the ApoB LDLR-binding domain wasfused to the protein. To confirm that staining was specific forneurons, we used two neuron-specific markers, calbindin (Fig. 3)and NeuN (Fig. 4). Calbindin is a 28-kDa membrane-boundcalcium channel protein found primarily in the Purkinje cells ofthe cerebellum (13) and the interneurons of the cortex (14) and,to a lesser degree, in other regions of the CNS. NeuN is aneuron-specific nuclear protein present in nearly all neurons ofthe CNS, with the exception of the Purkinje cells of the cere-bellum (15, 16).

The recombinant proteins sGCmApoB and sGFPmApoB (greenin Fig. 3 C and G) were found throughout the Purkinje cell layer(red in Fig. 3 B and F) of the cerebellum but not in the adjacentgranular layer (blue in Fig. 3 A and E). This expression patterncorresponded with the expression of the LDLR (purple in Fig. 3 Dand H), which suggests that the LDLR is necessary for uptake of therecombinant protein in the Purkinje cells. Protein uptake wasrestricted to recombinant protein containing the ApoB LDLR,because the control sGCm was not taken up by cells in thecerebellum (Fig. 3 I–K). Recombinant protein (yellow arrows) wasobserved sporadically throughout the cerebellum but was notassociated with calbindin (Fig. 3J). This is probably staining of theprotein associated with capillaries of the BBB.

NeuN is a nuclear marker associated with the vast majority ofneurons of the CNS. The recombinant proteins sGCmApoB andsGFPmApoB (green in Fig. 4 C and G) costained with the NeuNneuronal marker (red in Fig. 4 B and F) as well as the LDLR(purple in Fig. 4 D and H). In contrast, no staining of the sGCmcontrol protein was observed with the NeuN neuronal protein(Fig. 4 I–K). Recombinant protein was observed throughout theCNS, with greater concentrations found in the cortex, striatum,and olfactory bulb and lower concentrations in the granular layerof the hippocampus and the cerebellum. Thus, addition of the

ApoB LDLR-binding domain facilitated uptake of the recom-binant protein in both NeuN and calbindin subsets of neurons,and this uptake was associated with the expression of LDLR.

To visualize the passage of the recombinant protein across theBBB, we stained CNS sections with glial fibrillary acidic protein(GFAP), a marker of astrocytes and lectin, a protein that bindsto the endothelial cells of the BBB (see pictorial representationin Fig. 5M). Because astrocytes are known to express the LDLR(17), we suspected that we would observe uptake of the recom-binant protein by GFAP-positive cells. The GFAP (red) cellstake up the recombinant proteins sGCmApoB and sGFPmApoB(green in Fig. 5 C and G). The endothelial cells are visualized inpurple (Fig. 5 D, H, and L), and the adjacent GFAP-positiveastrocytes on the CNS side of the BBB are visualized as red (Fig.5 B, F, and J). The addition of the ApoB LDLR-binding sequencefacilitates passage of the recombinant protein across the endo-thelial cells (purple) of the BBB because no protein can beobserved within the vessels or associated with the endothelialcells. This protein is then taken up by astrocytes, as can bedetermined in the merged picture (yellow in Fig. 5 A and E). Incontrast, the sGCm protein lacking the ApoB LDLR-bindingdomain can be observed within the vessel lumen but is notdirectly associated with the endothelial cells of the BBB nor withthe astrocytes located adjacent to the endothelial cells (Fig. 5IInset).

Binding of ApoB to the LDLR results in targeting of theprotein to the lysosome, where the LDLR is released andrecycled to the cell surface. To determine whether the recom-binant protein containing the ApoB LDLR-binding domainwould behave in a similar fashion, brain sections were stained forthe sGCmApoB (green in Fig. 6C) as well as the lysosomalmembrane marker lysosomal-associated membrane protein(LAMP)1 (red in Fig. 6 B and E). Recombinant sGCmApoBprotein appears in the characteristic punctate location across thewhole cytoplasm and costained with LAMP1 at a perinuclearlocation (Fig. 6A). This confirmed the targeting of the recom-binant protein to the lysosomes. No costaining was observed

Fig. 2. Immunofluorescence staining of recombinant protein in peripheraltissues of injected mice. Sections (20 �m) of liver (A, D, and G), spleen (B, E, andH), and lung (C, F, and I) were stained for recombinant protein with the mycepitope tag (green) and nuclei (blue). (A–C) Photographs of a mouse injectedwith the LV–sGCmApoB vector. (D–F) Photographs of a mouse injected withthe LV–sGCm vector (without the ApoB tag). (G–I) Photographs of a saline-injected mouse. All photographs were taken with a �40 objective.

Fig. 1. Schematic drawing of lentiviral vectors. The glucocerebrosidase [orenhanced GFP (eGFP)] gene was cloned with the preprotrypsin secretory signal(ss) at the N terminus, and the myc epitope tag was cloned at the C terminus.The ApoB LDLR-binding domain was cloned at the C terminus of these genes,generating the constructs LV–sGCmApoB and LV–sGFPmApoB. (A) These con-structs were cloned into the self-inactivating LV vector under the control ofthe chicken �-actin/globin (CAG) promoter and containing the central purinetract [poly-purine tract (PPT)] as well as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to aid in transduction and tran-scription of the LV vector. (B) As a control, the sGCm gene without the additionof the ApoB LDLR-binding domain was generated. The LV–sGCmApoB vectoris 6,190 bp, whereas the control LV–sGCm vector is 6,103 bp. The LV–sGFPmApoB vector is 5,360 bp.

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with sGCm protein lacking the ApoB LDLR-binding domain(Fig. 6 D–F).

Sections of the CNS that were stained for calbindin, NeuN, orGFAP were randomly examined blindly to determine the per-centage of cells that appeared to take up the recombinantprotein. Sections from mice that had been injected with theLV–sGCmApoB virus were observed over at least 10 fields(Table 1). Calbindin cells were counted in two separate regionsof the brain. In the cerebellum, the calbindin-positive cells(Purkinje cells) all appeared to take up the recombinant protein.

In contrast, only 55% of the calbindin-positive cells of the cortex(interneurons) appeared to take up the recombinant protein.Approximately 30% of NeuN-positive neurons took up therecombinant protein, with the greatest percentage occurring inthe striatum and the lowest percentage in the hippocampus,where little or no uptake was observed. Astrocytes (GFAP-positive cells) almost universally appeared to take up the protein,with 84% of the cells staining for the recombinant protein.

Fig. 3. Immunofluorescence staining of recombinant protein in the cerebellum of injected mice. Brain sections (40 �m) were stained for calbindin (red), mycepitope of the recombinant proteins (green), LDLR (purple), and nuclei (blue). (A–D) A representative section from a mouse injected with LV–sGCmApoB. (E–H)A representative section from a mouse injected with LV–sGFPmApoB. (I–L) A representative section from a mouse injected with LV–sGCm. White arrows indicaterepresentative calbindin-positive cells. Yellow arrows are areas of recombinant protein staining that do not correspond to calbindin-positive cells. (B Inset) A fullystained Purkinje cell illustrating the soma as well as the dendrites and axons. Photographs were taken with a �40 objective.

Fig. 4. Immunofluorescence staining of recombinant protein in the brains of injected mice. Brain sections (40 �m) were stained for NeuN (red), myc epitopeof the recombinant proteins (green), LDLR (purple), and nuclei (blue). (A–D) A representative section from a mouse injected with LV–sGCmApoB. (E–H) Arepresentative section from a mouse injected with LV–sGFPmApoB. (I–L) A representative section from a mouse injected with LV–sGCm. White arrows indicaterepresentative NeuN-positive cells. Photographs were taken with a �63 objective.

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DiscussionIn this report, we have described the delivery of proteins to theCNS by addition of the LDLR-binding domain of ApoB. De-livery of the lentivector expressing the recombinant protein byi.p. injection was sufficient to deliver protein to the CNS acrossthe BBB. This transport was specific to the protein with theApoB LDLR domain, because the control protein sGCm did notcross the BBB when delivered in the same manner. Becausedelivery of the lentivector by i.v. or i.p. injection was performed

with no appreciable difference in delivery efficiency, the tech-nically simpler i.p. delivery route was performed for all exper-iments. Although the ApoB LDLR sequence is 38 aa, the lengthdid not appear to greatly affect delivery or function of therecombinant protein. The ApoE LDLR-binding site containingamino acids 152–168 (18, 19) functioned similarly to the ApoBsequence used in this report (data not shown).

The BBB is an effective way to protect the brain from the manychemicals f lowing around the body and common infections, yetthe brain needs certain nutrients and essential molecules, such ascholesterol, for normal functioning. We have taken advantage ofthe mechanisms that allows the brain to import lipids from

Fig. 5. Immunofluorescence staining of recombinant protein in the endothelial cells in the brains of injected mice. Brain sections (40 �m) were stained for GFAP(red), myc epitope of the recombinant proteins (green), lectin (purple), and nuclei (blue). (A–D) A representative section from a mouse injected withLV–sGCmApoB. (E–H) A representative section from a mouse injected with LV–sGFPmApoB. (I–L) A representative section from a mouse injected with LV–sGCm.White arrows indicate representative cells that costain for the GFAP cell marker as well as the recombinant protein. (I Inset) An enlargement of the area markedby a white square. A diagram depicting the cells of the BBB is shown in M. Endothelial cells, with their tight junctions, form a barrier around the lumen of thebrain capillary. The astrocytic processes are directly adjacent to these endothelial cells. Photographs were taken with a �40 objective.

Fig. 6. Colocalization of sGCmApoB with LAMP1. Brain sections (40 �m)were stained for LAMP1 (red), myc-tagged recombinant protein (green), andnuclei (blue). (A–C) Cells from a mouse injected with LV–sGCmApoB. (D–F)Cells from a mouse injected with LV–sGCm. Photographs were taken with a�100 objective.

Table 1. Percentage of cells examined in the brain that werepositive for the recombinant protein

Marker Cells, n Double-labeled, %

CalbindinCerebellum 83/83 100Cortex 153/85 55

NeuN 524/156 30GFAP 155/130 84

Brain sections were examined under a confocal microscope for cell-type-specific staining and were photographed. The photographs were later exam-ined, and the total numbers of calbindin-, NeuN-, or GFAP-positive cells werecounted. Calbindin-positive neurons were separated into two groups, com-posed of those in the cerebellum and those in the cortex. Then the numbersof these cells that costained for the recombinant protein were counted. Thenumbers of cells are depicted as the total cells counted per cells positive for therecombinant protein, and these values determine the percentage of double-labeled cells.

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the blood stream. One of the mechanisms involves the use of theLDL family of receptors, which are expressed on most cells in thebody, including endothelial cells, which make up the BBB.Specificity of uptake of LDL complexes is determined by theapolipoprotein bound to the complex, such that ApoB bindsspecifically to the LDLR and megalin (7, 9). LDLR is foundprimarily in the liver and the adrenal gland and, to a lesser extent,in the brain, muscle, and lung (reviewed in ref. 10). Examinationof nonneuronal tissues showed elevated levels of the glucocere-brosidase enzyme in the liver, spleen, and muscle (data notshown) and little or no increased activity in the lung. Thisspecificity of tissue distribution closely mimics the expressionlevels of the LDLR. In an extension of these studies, we deliveredan ApoB fusion lysosomal enzyme to the CNS of knockout micewith the protocol described here (manuscript in preparation).We observed a 50% increase in enzyme activity in the CNS ofthese animals over control animals that received the lysosomalenzyme without the ApoB fusion. Thus, the ApoB LDLR-binding peptide can be used to deliver a protein to many tissuesof the body in addition to delivery to the CNS.

The use of the LDLR as a target for BBB transport will provemost useful in the treatment of lysosomal storage diseases thatinvolve neural degeneration, such as Gaucher’s disease. Currently,enzyme-replacement therapy is an effective treatment for thelysosomal storage diseases; however, there is no effective treatmentfor the neuronal degenerative component (20). The infused enzymeis unable to cross the BBB and, therefore, has no access to theaffected neurons and astrocytes. Delivery of a lentiviral vectorexpressing the fusion protein described in this report may proveeffective in treating the neuronal degenerative component of manymetabolic disorders, while, at the same time, still effectively treatingthe peripheral component of the disease. Preliminary results indi-cate that delivery of an ApoB-fused lysosomal enzyme can preventsome of the neuronal loss associated with mucopolysaccharidosesdiseases (B.J.S., unpublished results).

Staining of the liver and spleen indicated significant protein inthese two organs. This was further verified by glucocerebrosidaseenzyme assay of tissue lysates. Although the immunohistochemistryindicated recombinant protein in both of these tissues, we could notdifferentiate between cells that were transduced with the LV andexpressing the recombinant protein and those cells that had takenup the protein because of LDLR-mediated endocytosis. In contrast,the recombinant protein observed in the CNS could have comefrom only transcytosis of the protein across the BBB because theLV is not known to cross the BBB. In addition, only the proteinsfused to the ApoB LDLR-binding domain were observed inneurons or astrocytes. The localization of the recombinant proteinto specific regions or cell types within the brain appears to correlatewith the expression of the LDLR, as is most evident in thecerebellum. In this region, Purkinje cells stained positive for LDLRas well as the recombinant protein in contrast to the adjacentgranular layer that did not stain for the LDLR.

Although we chose to target the LDLR for transport of therecombinant proteins to the CNS, it may be useful to examine otherreceptors on the BBB for targeting. The LDLR, similar to thetransferrin receptor that is also expressed on the BBB, targetsbound proteins to the lysosome. Other receptors may be moreuseful in delivering soluble growth factors that would be effectiveat the extracellular level. Alternatively, addition of a peptide-cleavage sequence may be possible to release the therapeuticprotein from the targeting sequence once it has entered the CNS.In this instance, �- or �-secretase (21) may be attractive targets forreleasing the protein from its targeting peptide.

Previous attempts to target proteins for transport across theBBB have all relied on the same technology. An antibody againsta receptor expressed on the BBB is bound to the targeted proteinin vitro before i.v. delivery (22–24). This method allows deliveryof the targeted protein to neurons; however, it does not allow for

continuous delivery of the targeted protein without repeated i.v.injections. The system we have described here utilizes the liveras a depot organ to express and distribute the recombinantprotein for sustained delivery to neurons and astrocytes of theCNS. It remains to be seen whether the level of delivery from thelentivector can achieve physiological levels; however, the firststep in targeting these proteins without the need of intracranialinjection or BBB-disrupting drugs now appears possible.

MethodsCloning. The human glucocerebrosidase gene (American TypeCulture Collection, Manassas, VA) was cloned in frame into thepcDNA3.1–myc–His vector (Invitrogen, Carlsbad, CA), generatinga C-terminal myc–His-tagged protein. Amino acids 3371–3409 ofhuman ApoB were cloned into the BamHI and HindIII sites of thisplasmid, replacing the His tag. Finally, the secretory leader se-quence of preprotrypsin (pFLAG–CMV-1; Sigma, St. Louis, MO)was cloned in frame at the ApaI site at the N terminus to aid insecretion of the protein. This construct was designated pcDNA–sGCmApoB. A similar construct was generated lacking the ApoBsequence and was designated pcDNA–sGCm.

These constructs were cloned into the third generation self-inactivating LV vector (25), with the chicken �-actin promoterdriving expression producing the vectors LV–sGCm and LV–sGCmApoB (Fig. 1). A similar vector was generated with theenhanced GFP reporter gene in place of the glucocerebrosidasegene generating the vector LV–sGFPmApoB. LV vectors weregenerated from these constructs with the TAT-less system, asdescribed previously (25).

Immunohistochemistry. Approximately 1 � 109 infectious units ofeach virus, as determined by p24 ELISA (PerkinElmer Life Sci-ences, Boston, MA), were injected i.p. into mice. Fourteen daysafter vector delivery, mice were perfused transcardially via the leftventricle with ice-cold PBS, followed by 4% paraformaldehyde.Frozen brains were sectioned on a microtome at 40 �m along thesagittal plane, and sections were stored in tissue-collection buffer(2:2:1 glycerol/ethylene glycol/0.1 M phosphate buffer) at 4°C untilstaining. Sections from noninjected mice as well as the threeinjected groups of mice were stained with a mouse monoclonalanti-myc (Santa Cruz Biotechnology, Santa Cruz, CA) or rabbitpolyclonal anti-myc antibody (Santa Cruz Biotechnology) to visu-alize the sGCm, sGCmApoB, or sGFPmApoB proteins and goatanti-LDLR (Santa Cruz Biotechnology). Cell-specific staining wasperformed with rabbit anti-calbindin (Chemicon, Temecula, CA),mouse anti-NeuN, guinea pig anti-GFAP (Advanced Immuno-Chemical, White City, CA), or biotinylated tomato lectin (VectorLabs, Burlingame, CA). In addition, some sections were stained forthe lysosomal membrane marker protein LAMP1 (Santa CruzBiotechnology). These were visualized with Alexa Fluor secondaryantibodies or streptavidin (Molecular Probes, Eugene, OR). Allsections were counterstained with DAPI. CNS sections wereviewed under a Leica confocal microscope (Leica, Deerfield, IL)and photographed.

The liver, lung, and spleen were collected from the perfusedmice and embedded in OCT (Tissue Tek, Hatfield, PA). Theorgans were then sectioned on a cryostat at 20-�m thickness andstained with a rabbit anti-myc antibody (Santa Cruz Biotech-nology) to visualize recombinant protein. Sections were coun-terstained with ToPro3 (Molecular Probes) to visualize nuclearDNA and then photographed on a Zeiss confocal microscope(Zeiss, Oberkochen, Germany).

We thank Dr. F. H. Gage (The Salk Institute for Biological Studies) forencouragement, help, discussions, and the generous gifts of reagents. I.M.V.is an American Cancer Society Professor of Molecular Biology and issupported, in part, by grants from the National Institutes of Health and theH.N. and Frances C. Berger Foundation. B.J.S. was a George E. Hewitt

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Foundation for Medical Research fellow and was also supported by aNational Institutes of Health Training Grant.

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