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First published online July 19, 2007
Stem Cells Vol. 25 No. 11 November 2007, pp. 2910 -2918
doi:10.1634/stemcells.2007-0238; www.StemCells.com
© 2007 AlphaMed Press

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TECHNOLOGY DEVELOPMENT

Selective Targeting of Adenoviral Vectors to Neural Precursor Cells in the Hippocampus of Adult Mice: New Prospects for In Situ Gene Therapy

Anke Schmidta, Stefan J.-P. Haasb, Steve Hildebrandta, Johanna Scheibea, Birthe Eckhoffb, Tomás Raceka, Gerd Kempermannc, Andreas Wreeb, Brigitte M. Pützera

aDepartment of Vectorology and Experimental Gene Therapy, Biomedical Research Center, and
bInstitute of Anatomy, University of Rostock, Rostock, Germany;
cCenter for Regenerative Therapies, Dresden, Germany

Key Words. Neural precursor/stem cells • Gene therapy • Dentate gyrus • Adenoviral vectors • Phage display pNestin-green fluorescent protein transgenic mice

Correspondence: Brigitte M. Pützer, Ph.D., M.D., Department of Vector Technology and Experimental Gene Therapy, Biomedical Research Center, Schillingallee 69, D-18057 Rostock, Germany. Telephone: 49-381-4945066; Fax: 49-381-4945062; e-mail: brigitte.puetzer{at}med.uni-rostock.de

Received March 30, 2007; accepted for publication July 9, 2007.
First published online in STEM CELLS EXPRESS   July 19, 2007.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
The adult brain contains neural precursor cells (NPC) that are attracted to brain lesions, such as areas of neurodegeneration, ischemia, and cancer. This suggests that NPC engineered to promote lineage-specific differentiation or to express therapeutic genes might become a valuable tool for restorative cell therapy and for targeting therapeutic genes to diseased brain regions. Here we report the identification of NPC-specific ligands from phage display peptide libraries and show their potential to selectively direct adenovirus-mediated gene transfer to NPC in adult mice. Identified peptides mediated specific virus binding and internalization to cultured neurospheres. Importantly, peptide-mediated adenoviral vector infection was restricted to precursor cells in the hippocampal dentate gyrus of pNestin-green fluorescent protein transgenic or C57BL/6 mice. Our approach represents a novel method for specific manipulation of NPC in the adult brain and may have major implications for the use of precursor cells as therapeutic delivery vehicles in the central nervous system.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
The properties of neural precursor cells (NPC) in the adult brain make them potentially suitable for restorative cell replacement strategies, as well as delivery vehicles for gene therapy [1]. Precursor cells are found throughout the entire adult brain [2], but they generate neurons only in two neurogenic regions: the subgranular zone (SGZ) in the hippocampal dentate gyrus [3, 4] and the lateral wall of the lateral ventricles, the subventricular zone (SVZ) [5]. Irrespective of potential heterogeneities within this population that require further research, NPC are attracted to various brain lesions, including areas of neurodegeneration and brain malignancies. For example, they have shown tropism for gliomas and degenerating spinal cord motor neurons in amyotrophic lateral sclerosis transgenic mice [6, 7]. Failing adult hippocampal neurogenesis has been brought into connection with the pathogenesis of disorders as diverse as dementias, major depression, and temporal lobe epilepsy [8–10]. The potential for the treatment of central nervous system (CNS) disorders, including those affecting the hippocampus, has advanced tremendously with the ability to identify specific genes whose defect or absence is responsible for the particular pathological condition [11] and genes that control precursor cell differentiation [12]. Successful delivery of these therapeutic genes to precursor cells will provide a significant advancement in therapy for certain brain disorders. So far, experimental cell therapy for CNS disorders has been based on the transplantation of in vitro expanded and genetically engineered NPC. The normal course of NPC development and migration in vivo is controlled by the microenvironment in the neurogenic regions of the brain. Because culture conditions strongly influence the phenotype of cells, culture could markedly alter the cells' response to their environment when reintroduced in vivo. One promising option for making use of the therapeutic potential of endogenous NPC in the brain and most notably the hippocampus is the application of a safe and efficient gene delivery system. Currently, the most efficient and popular way of introducing genes into NPC is by means of lentiviral vectors [13]. The chief concerns about this approach are frequent transgene silencing in situ and potential activation of nearby oncogenes during transgene integration [14]. On the basis of these previous studies and their limitations in practice, our study concentrates on the improvement of adenoviral vector systems for NPC transduction by linking adenovirus to new precursor cell targeting ligands.

To identify peptide ligands that selectively bind NPC from adult mouse hippocampus, we used the phage display technology. Neural precursor cells in vitro were defined by their ability to self renew and to differentiate into glia and neurons [15]. Cultures of primary neurospheres established from the hippocampal area of adult C57BL/6 mice were incubated with a 7mer phage library that offers the possibility to identify small peptides. Neurospheres contain few true stem cells and show differentiation at the core, making them a heterogeneous cluster of cells [16]. To overcome this limitation, we used neurospheres at an early stage, when a large proportion of the cells are proliferative and express precursor cell marker nestin. Moreover, we designed a two-stage experiment: we first used cultured cells to identify peptides with presumed high specificity. We then tested these peptides in vivo to confirm their sensitivity with immunohistological tools and according to the criteria developed to identify precursor cells in the neurogenic regions of the adult hippocampus [10, 17].


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Cell Culture and Peptides
Neural precursor cells were isolated from adult C57BL/6 hippocampus (Charles River Laboratories, Wilmington, MA, http://www.criver.com) and expanded as previously described [15]. 293, H1299, Pan 02, and NIH 3T3 cell lines were obtained from American Type Culture Collection (Manassas, VA, http://www.atcc.org). Cell cultures were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Karlsruhe, Germany, http://www.lifetech.com) supplemented with 10% fetal calf serum (FCS) (Biochrom AG, Berlin, http://www.biochrom.de) and 1% penicillin G/streptomycin (Life Technologies) in 5% CO2 at 37°C. All synthetic peptides were synthesized and purified by high-performance liquid chromatography (Metabion, Martinsried, Germany, http://www.metabion.com).

Phage Display
A Ph.D.-7 Phage Display Library (New England Biolabs, Frankfurt, Germany, http://www.neb.com) was amplified, purified, and titered according to the manufacturer's protocol. Briefly, neural precursor cells were incubated with 1.5 x 1011 plaque-forming units (pfu) of input phage library for 4 hours at 4°C. Phage recovery was performed using biopanning and rapid analysis of selective interactive ligands optimization method [18]. The panning procedure was repeated three times. Single phage plaques were picked in a final step and analyzed.

Titration Assay
Phage binding of infected cells in vitro was quantified as described [19]. Recovered phages were incubated with competent bacteria (Escherichia coli ER2738), and phage titer was determined by counting pfu.

Immunofluorescence and Laser Scanning Microscopy
Cells were seeded on glass coverslips and fixed with 10% formaldehyde/phosphate-buffered saline (PBS). After incubation with 1.5 x 1011 pfu of selected phages for 8–24 hours at room temperature (RT), slides were washed and treated with 500 mM NaCl/50 mM glycine to remove weakly bound phages. Cells were permeabilized with PBS/0.2% Triton X-100 and blocked with PBS/1% bovine serum albumin (BSA) containing 10% FCS. Slides were washed twice with PBS/1% BSA and incubated with anti-M13 antibody (Amersham Biosciences, Freiburg, Germany, http://www.amersham.com) for 1 hour. After washing with PBS, the slides were incubated with appropriate secondary Alexa Fluor 546-conjugated anti-mouse antibody (Molecular Probes Inc., Karlsruhe, Germany, http://probes.invitrogen.com) and subjected to fluorescence-activated laser scanning microscopy using TCS SP2 AOBS (Leica, Wetzlar, Germany, http://www.leica.com) and LSM 410 (Carl Zeiss, Jena, Germany, http://www.zeiss.com).

Competition Assays
Phages displaying a QTRFLLH, VPTQSSG, or HTFEPGV motif were titrated, and 1 x 109 pfu/ml were incubated with 106 cells either alone or together with QTRFLLH, VPTQSSG, or HTFEPGV synthetic peptide for 1 hour. Cells were washed five times with PBS containing 1% BSA and 0.1% Tween 20. Phages were eluted by freeze-cut technique [18] and pelleted by centrifugation. The amount of bound phages was determined by counting pfu after bacterial infection. For analysis of peptide-mediated specific recombinant adenoviral (Ad) vector that expresses red fluorescent protein AdRFP.QTRFLLH or AdRFP.VPTQSSG binding, 5 x 105 adult NPC were infected with 107 pfu of polyethylene glycol-linked virus in the absence or presence of synthetic peptide (0.3–3.0 µg/ml). Virus-cell interaction was visualized by immunofluorescence and laser scanning microscopy.

Adenovirus Vector Production and Peptides
Adenovirus serotype 5-derived vectors Ad vector expressing green fluorescent protein (AdGFP) (wild-type capsid) and pAdTLY477RAE (with green fluorescent protein [GFP]-Luc [AdGFPL], fiber mutant, coxsackie-adenovirus receptor [CAR]-binding ablated) viruses have been described earlier [20, 21]. For generation of AdRFP, RFP cDNA from the pDsRed Monomer plasmid (Clontech, Saint-Germain-en-Laye, France, http://www.clontech.com) was inserted into the KpnI/NotI restriction sites of the pShuttleCMV (AdEasy system). Virus was generated by homologous recombination following cotransfection with pAdEasy1 in E. coli BJ5183. All viruses were propagated, purified, and titrated by standard methods. Synthetic peptides were covalently linked to Ad vectors by bifunctional polyethylene glycol (PEG) (Shearwater Polymers, Munich, Germany, http://www.swpolymers.com). Briefly, bifunctional PEG was added to the virus with 5% wt/vol. To allow coupling of PEG to the adenovirus surface, samples were incubated by stirring for 1 hour at RT [22]. Repeated CsCl gradient centrifugation was performed to separate unbound PEG from the PEGylated virus followed by dialysis overnight at 4°C. Peptides dissolved in PBS containing 5% sucrose to a final concentration of 10 mM were added to the PEGylated virus at a concentration of 1 mM and incubated by stirring for 4 hours at 4°C. Separation of unreacted peptide was achieved by dialysis overnight [23, 24].

Mouse Experiments
Young adult male C57BL/6 mice were obtained from Charles River Laboratories. Transgenic mice that express GFP under the control of the rat nestin gene regulatory region (referred to as pNestin-GFP mice) were generated by M. Yamaguchi (Department of Physiology, University of Tokyo, Tokyo) [25]. Mice were housed in a dedicated pathogen-free animal facility under a 12-hour light/dark cycle with free access to food and water. All animal procedures were conducted in accordance with the German guidelines and with approval of the local authorities. Adenoviral vectors were injected at a low dose of 105 to 106 pfu/mouse in a volume of 2 µl of PBS2+ into one of the regions containing NPC (SVZ, lateral ventricle [LV], and dentate gyrus of the hippocampus [GD]) of the adult C57BL/6 and pNestin-GFP mouse brain, respectively. Briefly, mice were anesthetized by intraperitoneal injection of ketamin (75 mg/kg) and rompun (5.8 mg/kg) and mounted in a mouse adaptor (Stoelting Co., Wood Dale, IL) fixed in a rat stereotaxic apparatus (Kopf, Tujunga, CA, http://www.kopfinstruments.com). The skull was opened with a dental drill, and Ad vectors were injected into the SVZ (2 µl; AP, +1.2 mm; L, –0.8 mm; DV, –2.4 mm [D], coordinates according to bregma) [26], the LV (3 µl; AP, ±0 mm; L, –0.8 mm; DV, –1.8 mm [D]), or the GD (2 µl; AP, –1.9 mm; L, –1.2 mm; DV, –1.8 mm [D]) by using a glass capillary with an outer diameter of approximately 70 µm mounted on a 5-µl Hamilton syringe.

Immunohistochemistry
After anesthesia with an overdose of pentobarbital-Na+ (45 mg/kg), 3 days after virus-injection (n = 3–5 mice per group), mice were perfused with 5 ml of isotonic NaCl solution, followed by 50 ml of 3.7% paraformaldehyde (dissolved in 0.1 M PBS, pH 7.4). Brains were immediately removed from the skull and postfixed overnight, followed by an incubation overnight in PBS containing 20% sucrose at 4°C; finally, they were frozen in isopentane (–50°C) and stored at –80°C. Brains were cut in 30-µm-thick sections using a cryostat. Sections were washed three times with PBS, blocked for 1 hour at RT (PBS with 3% BSA, 3% normal goat serum, and 0.05% Triton X-100), and incubated with primary antibody against nestin (rabbit polyclonal, 1:400; Hiss Diagnostic, Freiburg, Germany, http://www.hiss-dx.de) in PBS/1% BSA/0.025% Triton X-100 overnight at 4°C. After washing, the sections were incubated with Cy3-conjugated anti-rabbit (1:500; Dianova, Hamburg, Germany, http://www.dianova.de) secondary antibody. Brain sections were mounted onto SuperFrostPlus-coated glass slides (Menzel-Gläser, Braunschweig, Germany, http://www.menzel.de), air dried, covered with a fluorescence mounting medium, and subjected to fluorescence-activated laser scanning microscopy.

For detection of immunological responses, sections were stained with anti-IbaI antibody (rabbit polyclonal, 1:250; Wako, Neuss, Germany, http://www.wakochemicals.de) and appropriate secondary antibody (polyclonal rabbit IgG, 1:200; Vector Laboratories, Peterborough, U.K., http://www.vectorlabs.com) followed by immunoperoxidase detection with the Vectastain ABC Elite kit (Vector Laboratories) and diaminobenzidine substrate (Linaris, Wertheim, Germany, http://www.linaris.de) according to the manual. Sections were mounted onto gelatin-coated glass slides, dehydrated in graded alcohol concentrations, and coverslipped with DePeX mounting medium (Serva Electrophoresis, Heidelberg, Germany, http://www.serva.de).

Statistical Analysis
Quantitative analysis to determine the area occupied by cells immunoreactive with antibody against nestin and expressing GFP or RFP within single 30-µm brain sections was performed with the Bio-Imaging-Analyzer System (Fuji, Düsseldorf, Germany, http://www.fujifilm.de) using TINA software. Brain sections containing the region of Ad vector application (defined region in the dentate gyrus between granular and subgranular zone and the hilus) were used for the quantitative analysis. Student's t test was used to determine the degree of statistical significance between values from different experimental groups.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Identification and Characterization of NPC-Specific Peptides
Biopanning was repeated three times to enrich the pool of phages in favor of the tightest binding sequence, resulting in more than 130 putative ligands after the third round. These candidates were preselected by comparing their binding efficiency for nestin-positive neurospheres in vitro using a titration assay (Fig. 1A). Phage clones 2, 82, and 119 demonstrated the strongest binding to NPC (10- to 20-fold stronger) compared with all others (clones 1, 3, 4, and 84 are shown as representative phages with significantly lower binding capacity). Subsequent analysis of the different phage clones for their peptide encoding sequence revealed at least 60 different sequences, which varied in their frequency (onefold to fourfold) and their internal motifs (supplemental online Table 1). Peptide sequences from the selected phages 2 (TPIQDYT), 82 (QTRFLLH), and 119 (VPTQSSG) were found 2–3 times. One sequence, QTFTRMY, appeared four times. Other peptides formed clusters depending on their motifs, for example, QT, SS, SL, and PL.


Figure 1
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  		Figure 1. Neural precursor cell specificity of preselected phages in vitro. (A): The binding efficiency of phages recovered after a third round of biopanning on cultured neurospheres was tested by titration assay. Precursor cells isolated from adult mouse brains were incubated with the indicated phage clones for 4 hours, including the best binding phages (phages 2, 82, and 119) compared with a representative choice of lower-binding phages (phages 1, 3, 4, and 84). The phage titer was determined after incubation of eluted phages with E. coli (ER2738) by counting pfu. (B): Titration results of phage clones 82 (QTRFLLH; top panel) and 119 (VPTQSSG; bottom panel) after binding to neurospheres and different other murine (Pan 02, NIH 3T3) or human (H1299, 293) cell lines. Columns show SEM from three independent experiments plated in duplicate. (A) and bottom panel of (B), *, p ≤ .005; top panel of (B), *, p ≤ .01. (C): Effect of synthetic peptides on phage binding to adult neural precursor cells. Neurospheres were incubated with phages in the absence or presence of indicated concentrations of specific QTRFLLH or VPTQSSG peptides for 1 hour. Unspecific HTFEPGV peptide was used as a control. The binding efficiency was determined by titration. (D): Immunofluorescent microscopy of cultured neurospheres incubated with the wild-type (DI), QTRFLLH (DII), VPTQSSG (DIII), or unspecific control phage 4 (DIV) for 24 hours. Phage binding and internalization was visualized using monoclonal antibody against M13. Scale bars = 20 µm ([DI, DIII, DIV], right column) and 200 µm ([DII], right column). Nuclei of single cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (left lane). Scale bars = 20 µm ([DI–DIV], left column). Perinuclear red fluorescence is indicative of internalized phages. (E): Immunofluorescent microscopy of neurospheres infected with PEGylated adenoviral vector that expresses red fluorescent protein AdRFP.QTRFLLH (EI, EII) or AdRFP.VPTQSSG (EIII, EIV) in the absence (EI, EIII) or presence (EII, EIV) of 3 µg/ml specific synthetic peptide. RFP expression was detected at 4 days after infection. Inset: neurospheres were counterstained with DAPI for visualization of cell numbers. Scale bars = 200 µm. Abbreviations: NPC, neural precursor cells; pfu, plaque-forming units.

 
To further determine the specificity of the identified phages 2, 82, and 119 for NPC, we examined their binding efficiency on additional cell lines of murine (Pan 02, NIH 3T3) and human (H1299, 293) origin. Among these, phages containing the sequence QTRFLLH (Fig. 1B, top panel) and VPTQSSG (Fig. 1B, bottom panel) exhibited the strongest binding activity to cultured NPC compared with other cell lines. Phage-cell interaction of the best binding QTRFLLH phage to NPC was 2.5 times stronger than to Pan 02 pancreatic cancer cells (1 x 108 pfu/ml) and to NIH 3T3 fibroblasts (9 x 107 pfu/ml), 83 times stronger than to H1299 lung cancer cells (3 x 106 pfu/ml), and 1,250 times stronger than to 293 human embryonic kidney cells (2 x 105 pfu/ml). Although the affinity of the QTRFLLH phage to bind precursor cells was found to be higher, the VPTQSSG phage bound them more selectively (binding of VPTQSSG to non-NPC was up to 10 times lower; Fig. 1B, bottom panel). The binding of both phages to NPC was dose-dependently inhibited in the presence of synthetic peptides, indicating that phage-precursor cell interaction strictly relied on the displayed peptide. We measured a 50% reduction of plaque-forming units by addition of 0.3 µg of synthetic peptides compared with an unspecific control peptide (Fig. 1C).

Selective Targeting of Labeled Ad Vectors to Hippocampal NPC in Adult Mice
Peptide-mediated internalization of a potential gene delivery system is a prerequisite for efficient transgene expression in target cells. To test both peptides for their capability to communicate this cellular uptake, neurospheres were seeded on glass coverslips and exposed to 1.5 x 1011 pfu/ml purified QTRFLLH or VPTQSSG phage for 8 hours. Phages released into the cell upon internalization were detected with anti-M13 antibody that binds to the phage coat protein. Both QTRFLLH and VPTQSSG peptides induced a strong uptake by NPC as indicated by their M13 antibody reactivity (Fig. 1D). Infection of cells with the most commonly used adenovirus serotype 5 is mediated via two cell surface receptors: first, the fiber protein binds to the CAR [27], and then, virus internalization is mediated through an interaction between an arginine-glycine-aspartate (RGD) sequence on the adenovirus penton base protein and cell surface {alpha}{nu}β3 and {alpha}{nu}β5 integrins [28]. However, we previously reported a complete lack of CAR and {alpha}{nu} integrin expression on adult NPC, indicating their resistance to adenoviral infection [15]. To determine whether the QTRFLLH and VPTQSSG peptides can mediate virus binding and internalization by NPC in vitro, we covalently linked both peptides to an Ad vector (wild-type capsid) that expresses the red fluorescent protein (RFP) and examined their binding specificity in infected NPC after adding 3.0 µg/ml synthetic QTRFLLH or VPTQSSG peptide (Fig. 1E). Unaltered RFP expression of AdRFP.QTRF-LLH- and AdRFP.VPTQSSG-infected neurospheres was observed by immunofluorescent microscopy in the presence of unspecific peptide but could not be detected in combination with the corresponding specific synthetic peptides. This supports the notion that the peptide ligands are similarly efficient in mediating adenovirus binding and infection of precursor cells in vitro.

Recombinant adenoviral vectors have been reported most efficient for delivering genes into nondividing cells such as glial cells and neurons [29]. A critical assumption for using the peptide-targeted Ad vector as a gene delivery system for endogenous NPC in the brain is, however, that it will not transduce other CNS cells. To test the targeting potential of the identified peptides under in vivo conditions, we injected 105 to 106 infectious viral particle (pfu/mouse) of AdRFP.QTRFLLH and AdRFP.VPTQSSG into the hippocampal area of young adult pNestin-GFP transgenic mice (Fig. 2A) that express GFP under the control of the nestin gene regulatory region shown to be essential for nestin expression in NPC [30]. In adult mice, GFP fluorescence was observed in regions where endogenous nestin is expressed and neurogenesis continues throughout life, such as SVZ, rostral migratory stream, olfactory bulb, and dentate gyrus (DG) (Fig. 2B) [25]. Fluorescence-activated laser scanning microscopy of brain sections from animals receiving AdRFP vectors that display the precursor cell targeting peptides revealed a strong specific labeling of GFP expressing cells in the SGZ (Fig. 2CI/CII, CIII/CIV, confocal xy-/xz-/yz-planes, right panels). By contrast, RFP-positive cells were not detectable in the respective region of the adult dentate gyrus after injection of the AdRFP.HTFEPGV virus carrying an unspecific control peptide (Fig. 2CV). A larger spectrum of CNS cells, especially with a neuronal and astroglial morphology, but no NPC were transduced by the wild-type AdRFP vector via CAR-binding (Fig. 2CVI). However, as the targeted adenoviruses with selected peptides still express intact fiber and penton base, they are still able to transduce other CNS cells, such as hippocampal parenchymal cells, as do the controls in Figure 2CV and CVI. This is apparent for AdRFP.QTRFLLH (Fig. 2C, top row), where the red fluorescence in regions where NPC are not is attributed to this double tropism. Quantitative analysis of single-labeled (GFP-positive) and double-labeled (GFP/RFP-positive) cells within various brain sections containing the dentate gyrus (region of Ad vector injection) revealed for the peptide tagged viruses 83.5% ± 9.4% (QTRFLLH) and 85.6% ± 4.4% (VPTQSSG) colabeled cells, whereas 15.5% ± 1% and 8.6% ± 1.7% were determined for the wild-type vector and the virus containing an unspecific control peptide (Fig. 2D).


Figure 2
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  		Figure 2. Capability of the QTRFLLH and VPTQSSG peptides linked to adenovirus to target adult neural precursor cells in vivo. (A): Nissl-stained coronal section of a pNestin-green fluorescent protein (GFP) transgenic mouse brain hemisphere through the region of the dorsal hippocampus containing the CA and the DG. Other principal structures (Co, CP, Th, and 3V) are indicated. The outlined area of the DG containing the population of neural precursor cells is shown in detail in (B). (B): Parallel section through the DG clearly shows that the nestin-containing somata of the GFP-expressing neural precursor cells are confined to the subgranular zone located between the h and the granule cell layer (arrows). The radial glia-like morphology of the so called type 1 cells is obvious (green fluorescence). The site of adenovirus injection for in vivo stem cell targeting is indicated by a star. (C): Fluorescence-activated laser scanning microscopy of the adult DG from pNestin-GFP transgenic mice after injection of AdRFP.QTRFLLH (CI) and AdRFP.VPTQSSG (CIII). In both cases, high numbers of transduced cells with a type 1 cell morphology could be detected (red fluorescence). In the images merged for RFP (red fluorescence) and GFP expression in nestin-containing precursor cells (green fluorescence) the colocalization is obvious (yellow fluorescence) (CII, CIV). Examples of dual-expressing (double staining) and non-dual-expressing (single marker) cells are indicated by arrows (white and black, respectively). AdRFP.HTFEPGV with an unspecific peptide (CV) and AdRFP (wild-type capsid [CVI]) were used as controls. Colocalization of GFP (nestin-positive)/RFP-expressing cells is also demonstrated by confocal xy-, xz-, and yz-planes (right panels). Scale bars = 500 µm (A), 100 µm (B, C). (D): Quantification of single- and double-labeled cells in 30-µm brain sections 3 days after adenoviral vector injection. Bars show SEM from each experimental group (*, p ≤ .01; **, p ≤ .005). Abbreviations: 3V, third ventricle; AdRFP, adenoviral vector that expresses red fluorescent protein; CA, cornu ammonis; Co, cortex; CP, cerebral peduncle; DG, dentate gyrus; h, hilus; Th, thalamus.

 
In addition, we examined the NPC-specific binding properties of the QTRFLLH and VPTQSSG peptides in context of an adenovirus fiber mutant with completely ablated native tropism. For this purpose, the peptides were covalently linked to the capsid-mutated adenovirus AdGFPL by bifunctional PEG. PEGylated AdGFPL.QTRFLLH (and AdGFPL.VPTQSSG) or AdGFPL.HTFEPGV, or AdGFP, the vector that contains a wild-type capsid, was subsequently injected into DG, SVZ, and LV of adult C57BL/6 mice. Brain sections were subjected to fluorescence-activated laser scanning microscopy. The mutation of both the CAR-binding domain of the fiber and the integrin-binding motif within the penton base of adenoviruses tagged with the selective peptides resulted in a specific staining of DG regions homing NPC as identified by the NPC-specific marker nestin (Fig. 3A–3F, 3N, 3O). A detailed inspection of single colabeled type 1 precursor cells at a higher magnification shows their radial glia-like morphology (Fig. 3G–3I). Moreover, type 2 cells known as transiently amplifying precursor cells, which develop from type 1 cells, are located in the subgranular zone (Fig. 3G–3I). Quantitative analysis of the area occupied by cells immunoreactive with antibody against nestin and expressing GFP in brain sections yielded 84.8% ± 9.1% and 82.5% ± 9.2% colabeled cells for the QTRFLLH and VPTQSSG vector, respectively. Additional nestin-positive cells visible in non-stem cell regions of the adult brain (not transduced by the specific vectors) are indicative for reactive astrocytes, in which nestin expression is reinduced following mechanical lesion [31]. Conversely, GFP expression was either not detectable by an Ad vector with the unspecific control peptide (Fig. 3J) or much wider after injection of the wild-type capsid AdGFP virus transducing several non-NPC cell types, such as astrocytes and neurons, in the adult GD (Fig. 3K, 3L). Importantly, however, GFP expression by AdGFPL.QTRFLLH was restricted to precursor cells located in dentate gyrus and was not detected in other injected brain regions, such as SVZ and the ependyma of the lateral ventricle (Fig. 3M). After injection, significant expression of fluorescent proteins (RFP and GFP) in NPC of the adult brain was detected over a period of 3 weeks (data not shown).


Figure 3
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  		Figure 3. Selective transduction of neural precursor cells in the adult C57BL/6 mouse brain by capsid-mutated AdGFPL.QTRFLLH and AdGFPL.VPTQSSG vectors. PEGylated AdGFPL.QTRFLLH (A–C, G–I), AdGFPL.VPTQSSG (D–F), AdGFPL.HTFEPGV (J), or AdGFP (K, L) was injected into the dentate gyrus of adult C57BL/6 mice. Brain sections were analyzed by immunohistochemistry. (G): AdGFLP.QTRFLLH-infected GFP-expressing (green fluorescence) type 1 (white arrow) or type 2 (yellow arrow) precursor cells are indicated (representative of both viruses tagged with specific peptide). The radial glia-like morphology of the type 1/2 cells and the location of their somata in the subgranular zone are shown. Shown are nestin (red fluorescence [B, E, H]) and merge (yellow [C, F, I]). (J): AdGFPL.HTFEPGV failed to transduce cells in the dentate gyrus. (K, L): Cells with a neuronal or a mature astrocyte morphology were infected by AdGFP. (M): GFP-positive cells were not detected after injection of AdGFPL.QTRFLLH in the SVZ. Scale bars = 100 µm. (N, O): Double-labeled cells are shown by confocal microscopy (xy-/xz-/yz-planes). Abbreviations: AdGFP, adenoviral vector that expresses green fluorescent protein; AdGFPL, adenoviral vector that expresses green fluorescent protein-Luc; gcl, granular cell layer; h, hilus; SVZ, subventricular zone.

 
With respect to the immunogenic properties of adenovirus, we analyzed the inflammatory responses of the various injected constructs in the brain using IbaI antibody (mouse macrophages and activated microglial cells). As shown in Figure 4, all vectors elicited a similar moderate IbaI immunoreactivity. Our findings are consistent with previous data demonstrating that although ablation of CAR and integrin binding almost completely eliminates Ad vector-mediated brain transduction, fully ablated viruses elicit inflammation indistinguishable from that induced by wild-type vectors [32]. The results indicate that brain inflammation occurs independently of adenovirus binding and infection of cells via CAR and integrin receptors. In addition, a defined immunoreactivity was also visible after injection of saline (Fig. 4E), suggesting that mechanical stress induced by the needle per se contributes to a positive immune response.


Figure 4
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  		Figure 4. Specific peptide-tagged and wild-type adenoviral constructs elicit similar moderate inflammatory responses in the brain. Anti-IbaI immunohistochemistry of brain sections from mice injected with AdGFPL.QTRFLLH (A), AdGFPL.VPTQSSG (B), AdGFPL.HTFEPGV (C), and AdRFP wild-type Ad vector (D). (E): Saline was injected as control. Scale bars = 100 µm. Abbreviation: AdRFP, adenoviral vector that expresses red fluorescent protein.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
In summary, we intended to target antigens on NPC by a genetically modified adenovirus vector linked to the antigen-binding peptide. We carried out phage displays using endogenous neurospheres isolated from the hippocampus of adult mice to identify peptides that selectively bind NPC. Thereby, two peptide ligands were identified that mediated good phage binding and internalization to cultured neurospheres.

Our aim was to demonstrate the usefulness of the identified peptides in targeting adenovirus-mediated gene delivery to NPC in vivo using an Ad vector with the wild-type capsid and a CAR-binding ablated virus in normal and pNestin-GFP transgenic mice. Analysis of Ad vector distribution after virus injection into the adult hippocampus, known to home precursor cell populations, demonstrated a highly specific infection of type 1 and type 2 precursor cells in the granule cell layer of the hippocampal dentate gyrus by the capsid-mutated Ad vector displaying the specific peptides. Apart from the notable target cell specificity of AdGFPL.QTRFLLH, GFP expression in precursor cells was confined to dentate gyrus, whereas other injected regions, such as the subventricular zone and the ependyma of the lateral ventricle, were negative. Our data suggest that intrinsic differences in precursor cells between different brain regions can translate into a diverse spectrum of cell-surface receptors on these cells. This indirect indication of precursor cell heterogeneity between the hippocampus and other brain regions is in accordance with previous reports by others [33, 34]. Consequently, our findings imply that NPC-specific Ad vectors will needed for specific brain regions. Moreover, the cell type binding assays showed less binding to human cells, whereas the two mouse cell lines had only slightly lower binding than mouse NPC, which could be indicative of a possible species difference. Whether the identified peptides bind human NPC cannot be answered definitively at this point. Support that these peptides indeed bind human NPC, however, comes from our previous study using the same phage library, where a tumor-specific peptide was identified in transgenic mice that also mediates binding to human cells in vitro and in vivo [35].

The entire nature of NPC is that they divide, and they do so quite rapidly for variable periods of time. Unlike the case of a retro- or lentivirus, each round of proliferation should therefore dilute the viral particles to some degree. Thus, duration of transgene expression after adenovirus injection into the brain is an important issue. With the first-generation Ad vector used in this study, we found a significant labeling of NPC in the adult brain over a period of 3 weeks, demonstrating that this approach/vector type is useful for most types of experiments. In fact, short-term expression is not a limitation of first-generation Ad vectors administered into the brain. As shown by others they can express at stable levels for at least 6 months [36], and behavioral recoveries for up to 4 months have been described previously [37]. However, during the lifetime of an individual previously subjected to gene transfer with an adenovirus, an immune challenge by natural infection with the same or a closely related serotype could be a likely occurrence that leads to vector instability in CNS cells. We observed a similar immunoreactivity for the specific peptide-tagged and wild-type vectors, supporting the notion that adenovirus-mediated brain inflammation occurs independently of virus binding and infection of cells via CAR and integrin receptors [32]. To achieve long-term gene delivery in the absence of inflammatory responses, high-capacity adenoviral vectors (HC-Ad), either genetically modified or covalently linked to the peptides that confer specific vector retargeting to NPC, can be used. New generation HC-Ad vectors that are deleted of all viral genes are completely immune to antiadenoviral immune response and have been shown to succeed in maintaining stable and long-term transgene expression in the brain [36].

In general, our results support the use of Ad vector systems specifically targeted to adult NPC as a perfect tool to efficiently manipulate these cells either by direct injection in the brain or systemic vector application. This will allow to benefit of their potential to serve as a source for cell replacement and as delivery system for therapeutic genes to treat various CNS disorders.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
We thank Anja Stoll for technical assistance and Frank Lüthen for support in confocal microscopy. We are grateful to Masahiro Yamaguchi (University of Tokyo) for authorization to use the pNestin-GFP mice and to Ramon Alemany (University of Alabama at Birmingham) and Renata Pasqualini (University of Texas) for making reagents available. This work was supported by Grant 01ZZ0108 from Bundesministerium für Bildung und Forschung and the Medical Faculty of Rostock University.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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