In vivo targeting of subventricular zone astrocytes

Progress in Neurobiology 92 (2010) 19–32

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Progress in Neurobiology journal homepage: www.elsevier.com/locate/pneurobio

In vivo targeting of subventricular zone astrocytes Carlyn Mamber a,*, Joost Verhaagen b, Elly M. Hol a,** a

Astrocyte Biology & Neurodegeneration, Netherlands Institute for Neuroscience, An Institute of the Royal Netherlands Academy of Arts and Sciences, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands b Neuroregeneration, Netherlands Institute for Neuroscience, An Institute of the Royal Netherlands Academy of Arts and Sciences, Meibergdreef 47. 1105 BA Amsterdam, The Netherlands

A R T I C L E I N F O

A B S T R A C T

Article history: Received 12 February 2010 Received in revised form 20 April 2010 Accepted 27 April 2010

The subventricular zone (SVZ) is a dynamic cellular niche with unique neurogenic properties that are, as of yet, not fully understood. Astrocytes residing in the SVZ have been shown to spawn migratory neuroblasts via transitory amplifying progenitor cells. These migratory neuroblasts play a role in maintaining the olfactory circuitry in healthy brains and potentially have restorative properties after brain injury. Therefore, it is imperative to understand the basic nature of these neurogenic astrocytes in order to gain a more cohesive picture of SVZ adult neurogenesis. However, one of the obstacles in this line of research is to specifically genetically modify SVZ astrocytes. Viral vector systems, based on adeno-associated viruses and lentiviruses, are flexible gene transfer systems that allow long-term transgene expression in a host cell. Electroporation allows for the transient expression of larger transgenes; whereas the cre/loxP system provides a lifetime of inherently stable genetic modulation. The benefits and drawbacks of these transduction methods and the application of various astrocyte-specific promoters are discussed with regard to their efficiency and accuracy when transducing adult SVZ astrocytes in the mouse brain. In vivo studies that manipulate gene expression in SVZ astrocytes will be essential to fully dissect and understand the complex molecular and cellular properties of the SVZ in the upcoming years. ß 2010 Elsevier Ltd. All rights reserved.

Keywords: AAV Astrocyte Cre/LoxP Electroporation Lentivirus Neural stem cell Subventricular zone

Contents 1. 2.

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The neurogenic system in rodent . . . . . . . . . . . . . . . . . . . . . . . . . . . Adeno-associated viral vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. AAV4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. AAV5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. AAV9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Comparison of AAV4, AAV5, and AAV9 . . . . . . . . . . . . . . . . . Lentiviral vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. VSV lentiviral envelope. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. LCMV lentiviral envelope . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Mokola lentiviral envelope . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Comparison of VSV, LCMV, and Moloka lentiviral envelopes Electroporation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: AAV, adeno-associated viruses; ALDH1L1, aldehyde dehydrogenase 1 family member L1; AON, anti-sense oligonucleotide; ApoE, apolipoprotein E; Aqp4, aquaporin 4; Ara-C, cytosine arabinoside; CMV, cytomegalovirus; Cx30, connexin 30; DCX, doublecortin; Dlx2, distalless homeobox 2; EGFR, epidermal growth factor receptor; EIAV, equine infectious anemia virus; EP, electroporation; FIV, feline immunodeficiency virus; GFAP, glial fibrillary acid protein; GFAPd, glial fibrillary acid proteindelta; GLAST, glutamate aspartate transporter; GLT-1, glutamate transporter-1; HIV, human immunodeficiency virus; Id1, inhibitor of DNA binding 1; ITR, inverted terminal repeat; LCMV, lymphocytic choriomeningitis virus; LeX, Lewis X; LV, lentivirus; Mash1, achaete-scute complex homolog 1; mCD24, mouse cluster of differentiation 24; Mcm2, minichromosome maintenance 2; OB, olfactory bulb; PCNA, proliferating cell nuclear antigen; PDGFR, platelet derived growth factor receptor; PSA-NCAM, polysialylated neural cell adhesion molecule; RMS, rostral migratory stream; SIV, simian immunodeficiency virus; Sox2, sex determining region Y-box 2; SVZ, subventricular zone; TuJ1, neuron-specific class III beta-tubulin; Vim, vimentin; VSV, vesicular stomatitis virus. * Corresponding author. Tel.: +31 20 5665508. ** Corresponding author. Tel.: +31 20 5665498. E-mail addresses: [email protected] (C. Mamber), [email protected] (E.M. Hol). 0301-0082/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.pneurobio.2010.04.007

C. Mamber et al. / Progress in Neurobiology 92 (2010) 19–32

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Promoter choice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. The glial fibrillary acid protein promoters. . . . . . . . . . . . . . . . . . . . . . . . . 5.2. The aldehyde dehydrogenase 1 family member L1 (ALDH1L1) promoter 5.3. The glutamate aspartate transporter (GLAST) promoter . . . . . . . . . . . . . . 5.4. Astrocyte-specific promoter comparisons . . . . . . . . . . . . . . . . . . . . . . . . . Cre/LoxP technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. The neurogenic system in rodent Deep in the brain, wrapping the lateral ventricle, lies the subventricular zone (SVZ). The SVZ is a dynamic place, serving as the most predominant source of adult neurogenesis (Doetsch and Alvarez-Buylla, 1996). In 1912, Allen was the first to make the observation that ‘‘after the dividing cells have disappeared from all other areas [of the brain] they are still to be found in the limited zone along the lateral ventricles of the cerebrum’’ (Allen, 1912). Present in mammals (Doetsch et al., 1999; Sanai et al., 2004), the SVZ seems as though it is a preserved relic left behind from the developmental brain. It forms under the ventricular zone during development and slowly reduces in size leaving a small, highly proliferative layer in the adult brain (Conover and Allen, 2002). The mouse SVZ has a distinctive cellular architecture (Fig. 1) composed of four main cell types. A monolayer of ependymal cells (also known as type E cells) lines the ventricular surface. SVZ astrocytes (type B cells) cover the top of this ependymal monolayer and sometimes extend a single cilium reaching into the lateral ventricle (Zhu and Dahlstrom, 2007). Other SVZ astrocytes encase neuroblasts (sometimes referred to as type A cells), physically separating them from the striatum. Scattered throughout the SVZ are clusters of transitory amplifying progenitor cells (type C cells) characterized by their large globular morphology and reticulated nucleoli (Doetsch et al., 1997; Garcia-Verdugo et al., 1998). Over 30,000 neuroblasts exit the mouse SVZ each day and travel anteriorly at speeds around 122 mm/h to the olfactory bulb (OB;

[(Fig._1)TD$IG]

Fig. 1. Mouse subventricular zone and rostral migratory stream. (A) Ependymal cells (green) act as a border between the SVZ and the lateral ventricle. SVZ astrocytes (blue) encase neuroblasts (yellow) and are sometimes observed to extend a single cilium contacting the lateral ventricle. The largest cell type in the SVZ is the transitory amplifying cell (pink). Neuroblasts migrate through astrocytelined tunnels in the RMS to reach the OB where they become functionally integrated into either the granular or periglomerular layer as interneurons. (B) SVZ astrocytes (blue) produce transitory amplifying progenitor cells (pink), which, in turn, give rise to neuroblasts (yellow). Key: LV—lateral ventricle; OB—olfactory bulb; RMS—rostral migratory stream; SVZ—subventricular zone.

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Alvarez-Buylla et al., 2001; Garcia-Verdugo et al., 1998; Wichterle et al., 1997). This pathway known as the rostral migratory stream (RMS; Fig. 1), consists of astrocytes encapsulating migratory neuroblasts that travel from the SVZ to the OB (Luskin, 1993), a distance of up to 5 millimeters in rodents and 2 centimeters in primates (Kornack and Rakic, 2001; Lois et al., 1996). Neuroblasts accomplish this feat by ‘‘literally sliding over their neighbors’’, a processes called chain migration (Wichterle et al., 1997). Once in the OB, neuroblasts amalgamate into the granule or periglomerular cell layer. There, neuroblasts mature into two main classes of interneurons, granular cells and periglomerular cells and become an integral part of OB circuitry (as reviewed by Lledo et al., 2008; Whitman and Greer, 2009). These new granule and periglomerular neurons are thought to play an important role in olfactory discrimination, sexual behavior, and maternal responses (AlvarezBuylla and Garcia-Verdugo, 2002; Whitman and Greer, 2009). Moreover after cerebral injury and stroke, neuroblasts are found to migrate via the adult correlate of the lateral cortical stream to the lesion site and differentiate into functional neurons (Jin et al., 2003; Yamashita et al., 2006). Combined, these results demonstrate the importance of neuroblasts in both the healthy and injured brain. Where do these neuroblasts originate from? An eloquent study in 1999 by the laboratory of Alvarez-Buylla helped to answer this question. They infused an anti-mitotic agent into the brains of 2–3 month old mice, resulting in the elimination of all rapidly proliferating cells, namely neuroblasts and transitory amplifying progenitor cells. Within a day of the cessation of the anti-mitotic treatment, glial fibrillary acidic protein (GFAP)-expressing astrocytes were found to divide producing transitory amplifying progenitor cells. These transitory amplifying progenitor cells then gave rise to neuroblasts. To see if neuroblasts were derived from astrocytes under normal physiological conditions, a reporter retrovirus containing b-galactosidase, targeting GFAP-positive dividing cells was injected into the lateral ventricle. 3.5 days after retrovirus injection, b-galactosidase positive cells were found in the SVZ and RMS. Within 2 weeks, these neuroblasts had integrated as interneurons into the OB. All in all, these results demonstrate that SVZ astrocytes directly produce highly proliferative transitory amplifying progenitor cells that eventually give rise to neuroblasts. These neuroblasts, then, migrate towards the OB via the RMS and are able to become functionally integrated within two weeks of their initial birth (Doetsch et al., 1999). Furthermore, this study in combination with a study from the Steindler laboratory, illustrating that SVZ astrocytes are able to form multipotent neurospheres well into adulthood, demonstrate that SVZ astrocytes are quiescent progenitor cells of the mature brain (Laywell et al., 2000). As more and more evidence points towards a heterogeneous SVZ astrocyte population, it is unclear what specific astrocyte phenotype gives rise to neuroblasts. In the rodent brain, there are two main types of SVZ astrocytes—B1 and B2 cells. B2 astrocytes separate neuroblasts from the striatum, while B1 astrocytes are located more ventrally, separating neuroblasts cells from the

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ependymal layer (Doetsch et al., 1997). Both B1 and B2 astrocytes contribute to the neurogenic environment of the SVZ (Lledo et al., 2006; Wang and Bordey, 2008), though B1 astrocytes seem to be the quiescent, slowly diving progenitors that give rise to neuroblasts in vivo (Doetsch et al., 1997; Pastrana et al., 2009). Like rodents, there is now evidence that humans may also have B1 and B2 astrocytes. Human B1 astrocytes are reported to have the largest nuclei of all the SVZ cell types, while human B2 astrocytes have elongated nuclei (Curtis et al., 2005). Since the exact characteristics of these human B1 and B2 astrocytes have yet to be fully described, it is difficult to conclude that the so-called B1 astrocytes in humans are the same quiescent, slowly diving progenitors as seen in rodents. Moreover, the presence of these B1 and B2 astrocytes has not been universally corroborated (Quinones-Hinojosa et al., 2006). This ambiguity stems from the lack of appropriate markers that distinguish B1 and B2 astrocytes. Furthermore, B1 and B2 astrocytes reside in close proximity to each other, so there are technical restraints that prevent the dissection of a single B1 astrocyte from its neighbor. There is a need for precise delineation of the exact attributes of B1 and B2 astrocytes in both human and rodent neurogenic systems in order to fully comprehend the human neurogenic system. That said, the human SVZ, like that of rodents contains GFAP-positive astrocytes that serve as progenitor cells, giving rise to neuroblasts (Sanai et al., 2004). It is still unknown whether all SVZ astrocytes possess innate neurogenic abilities or whether these abilities are restricted to a subpopulation of astrocytes. What are the underlying molecular mechanisms that dictate the neurogenic potential of these astrocytes? The structure of the intermediate filament network has come to light as conceivably being a major contributor to this observed neurogenic potential. The intermediate filament network of a neurogenic astrocyte is comprised of nestin, vimentin, and various GFAP isoforms (Gilyarov, 2008; Middeldorp et al., 2010; Roelofs et al., 2005). An isoform of GFAP, GFAPd, shows strong expression in human SVZ astrocyte stem cells (Roelofs et al., 2005; van den Berge et al., 2010). It is expressed in radial glia (Middeldorp et al., 2010) and has been implicated in glia proliferation (Martinian et al., 2009). Intermediate filaments have also been shown to play major roles in cell motility, cell migration, cell

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division, and cell survival (Lepekhin et al., 2001; Pallari and Eriksson, 2006). Other factors have also been linked with neurogenic astrocytes, such as the epidermal growth factor receptor, Notch1, and the helix-loop-helix transcription factor Id1 (Conover and Allen, 2002; Nam and Benezra, 2009; Pastrana et al., 2009). However, the exact characteristics of SVZ astrocyte subpopulations still remain, for the most part, ambiguous (see Table 1 for an overview of SVZ cell type markers). Further research is needed to gain a more complete picture of SVZ astrocytes and the processes governing them in vivo. This gained knowledge will allow for the development of new treatment avenues that can redirect endogenous brain repair in such cases as traumatic brain injury and neurodegenerative diseases. Endogenous gene manipulation and transgene delivery are ideal tools to investigate the fundamental nature of these astrocytes. Moreover, such delivery tools are ideal for in vivo use as they have the potential to specifically modulate transgene expression in a certain cell type. This specificity allows for the study of modulatory effects not only on the cellular level but also on the system, as a whole. There are many gene delivery systems currently available, each with their advantages and disadvantages. This paper aims to review the most promising transgene delivery and gene manipulation systems with a special focus on their probable efficacy and accuracy when transducing adult SVZ astrocytes. 2. Adeno-associated viral vectors Originally discovered in a simian adenovirus preparation (Atchison et al., 1965), Adeno-associated viruses (AAVs) offer a safe and efficient means of in vivo gene transfer (for recent review see Aucoin et al., 2008; Terzi and Zachariou, 2008). They belong to the genus Dependovirus, which denotes their inability to replicate, and therefore establish an active infection, without the presence of a helper-virus (Aucoin et al., 2008; Kaludov et al., 2001). These small (20–26 nm) viruses are non-toxic (Aucoin et al., 2008; McCown et al., 1996) and are not associated with any disease (Terzi and Zachariou, 2008). AAV is comprised of a 4.7 kb single strand of DNA encompassed by a non-enveloped capsid (Aucoin et al., 2008). Most inserted transgenes stay around this size; though there are

Table 1 SVZ cell type specific markers. SVZ cell type

Marker

Reference

Neuroblast

Doublecortin (DCX) Mouse Cluster of Differentiation 24 (mCD24) Polysialylated Neural Cell Adhesion Molecule (PSA-NCAM) Neuron-specific class III beta-tubulin (TuJ1)

Lledo et al. (2006) Calaora et al. (1996) Doetsch et al. (1997) Doetsch et al. (1997)

Astrocyte

Glial Fibrillary Acidic Protein (GFAP) Lewis X (LeX) Nestin S100b Sex Determining Region Y-Box 2 (Sox2) Vimentin (Vim)

Garcia-Verdugo et al. (1998), Garcia et al. (2004), Liu et al. (2006) Capela and Temple (2002), Platel et al. (2009) Doetsch et al. (1997) Platel et al. (2009), Raponi et al. (2007) Komitova and Eriksson (2004) Doetsch et al. (1997)

Astrocyte (B1)

Inhibitor of DNA Binding 1 (Id1HIGH)

Nam and Benezra (2009)

Transitory Amplifying Progenitor

Distalless Homeobox 2 (Dlx2) Epidermal Growth Factor Receptor (EGFR) Lewis X (LeX) Achaete-Scute Complex Homolog 1 (Mash1)

Doetsch et al. (2002) Doetsch et al. (2002), Platel et al. (2009) Capela and Temple (2002), Platel et al. (2009) Kuo et al. (2006), Nam and Benezra (2009)

Ependymal Cell

GFAP Noggin Nestin Vimentin

Doetsch et al. (2002) Lim et al. (2000) Garcia-Verdugo et al. (1998) Garcia-Verdugo et al. (1998)

Though the exact expression profiles of all the cell types in the SVZ remain incompletely understood, there are several known markers that help in cell type identification. It should also be noted here that markers such as Ki67, Minichromosome Maintenance 2 (Mcm2) and Proliferating Cell Nuclear Antigen (PCNA) are used to identify proliferating cells.

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reports of AAV vectors that package larger transgenes of up to 8.9 kb (Allocca et al., 2008). Caution must be taken when using larger transgenes in combination with AAVs, as most studies to date note that transgenes over 5.2 kb significantly reduce both packaging and transduction efficiency. Moreover with larger constructs, the risk of truncating the inserted transgene also considerably increases (Grieger and Samulski, 2005; Wu et al., 2009). Therefore, when designing an AAV construct, it is best to keep the maximal insert around 4.7–5.2 kb in length. Recombinant AAVs are able to integrate into the genome, albeit at a low rate. AAV particles show a maximum integration of 0.1–15% (McCarty et al., 2004) which occurs at random positions in the host’s genome (Young et al., 2000). Interestingly, AAV has been reported to integrate with a higher efficiency in quiescent hematopoietic stem cells due to selective pressure (Han et al., 2008; Paz et al., 2007). What remains unclear, however, is how these observations in hematopoietic stem cells will translate into the B1 quiescent astrocyte situation. It must be noted that even if there is a lack of integration, AAVs can still induce long lasting transgene expression ranging from nine months to six years (Belur et al., 2008; Haberman et al., 1998; Stieger et al., 2009; Zincarelli et al., 2008). Currently, there are over 100 known AAV serotypes (Kwon and Schaffer, 2008). These differ in protein composition of their capsids and, to a lesser extent, inverted terminal repeat (ITR) sequence (Aucoin et al., 2008; Kwon and Schaffer, 2008). Each of the serotypes has its own distinct tropism profile. To analyze serotype directed tropism, Zincarelli et al. (2008) cross-packaged nine AAV serotypes with a luciferase transgene flanked by AAV2 ITRs. These recombinant AAVs were injected into the tail vein of mice. The luciferase transgene expression was then characterized at different time-points. Zincarelli et al. (2008) found that each of the nine tested AAV serotypes displayed a unique pattern of both tissue tropism and level of luciferase expression; thus, demonstrating the importance that serotype capsid plays in targeting (Zincarelli et al., 2008). Out of all the AAV serotypes, only three have been shown to transduce astrocytes efficiently in vivo, namely AAV4, AAV5, and AAV9. AAV4 was originally isolated from a simian viral stock (Parks et al., 1967), while AAV5 and AAV9 are derived from human sources (Bantel-Schaal and zur Hausen, 1984; Gao et al., 2004). 2.1. AAV4 AAV4 has been shown to transduce ependymal cells and astrocytes in mice (Davidson et al., 2000; Liu et al., 2005). Although the exact mechanism of AAV4 transduction remains elusive, AAV4 binding is probably dependent upon the presence of an a2-3 sialic acid-containing glycoprotein (Kaludov et al., 2001). AAV4 injected into the anterior SVZ preferentially transduces GFAP-positive cells of the SVZ and RMS. Liu et al. (2005) propose that these GFAP-positive cells are the quiescent slowly diving SVZ astrocytes, due to their close proximity to migrating neuroblasts. However, a study by Davidson et al. (2000) reports that AAV4 can only transduce ependymal cells. Both groups used the same expression cassettes, but varied in the injection site as well as the age of mice. A comparison of these two studies illustrates that intraventricular or striatal injections of AAV4 transduce ependymal cells almost exclusively, while SVZ injections result in both ependymal and SVZ astrocyte transduction. The choice of injection site has been previously used to successfully target specific brain regions and cell types (Hommel et al., 2003). So the choice of the injection site must be kept in mind not only when characterizing tropism, but also when specific targeting is desired. Taken together, the current evidence indicates that AAV4, when injected into the adult SVZ, is able to transduce both ependymal cells and SVZ astrocytes.

2.2. AAV5 AAV5 has a wider tropism than AAV4. It is able to transduce ependymal cells, choroid plexus cells, neurons, oligodendrocytes, microglia, and astrocytes in mice (Davidson et al., 2000; Di Pasquale et al., 2003; Sevin et al., 2006; Watson et al., 2005). Like AAV4, AAV5 primarily transduces ependymal cells when injected intraventricularly (Davidson et al., 2000). Watson et al. (2005) hypothesize that this limited penetration occurs because AAV5 binds quickly upon injection, restricting much of the virus from entering the surrounding parenchyma. Injection of AAV5 into the cerebellum or internal capsule primarily transduced neurons (80%), followed by astrocytes (18%). AAV5 was also able to transduce microglia and oligodendrocytes to a much lesser extent (2%; Sevin et al., 2006). The wide tropism displayed by AAV5 may be explained by its receptor interactions. AAV5 interacts with the platelet derived growth factor receptor (PDGFR; Di Pasquale et al., 2003). The PDGFR is expressed by neurons, immature oligodendrocytes, and astrocytes (Besnard et al., 1987; Oumesmar et al., 1997; Wegner, 2008). Interestingly, cells of the SVZ also express PDGFR. These include nestin-positive and GFAP-positive cells, or more specifically the quiescent SVZ astrocytes (Ishii et al., 2008; Jackson et al., 2006). These data along with AAV5’s efficient binding ability suggest that AAV5 may be well suited to specifically transduce SVZ astrocytes, if directly injected into the SVZ. 2.3. AAV9 AAV9 has been recently reported to target adult astrocytes more robustly than its cousins AAV4 and AAV5 in mice and cats (Duque et al., 2009; Foust et al., 2009). Foust et al. (2009) found that AAV9 tail vein injection preferentially transduced adult astrocytes, over any other adult cell type. This astrocytic transduction preference following tail vein administration is still awaiting independent confirmation. Presumably, AAV9 binds to 37/67 kD laminin receptors (Akache et al., 2006) located on the perivascular end feet of astrocytes (Foust et al., 2009). It is not known, however, whether this robust astrocytic transduction would remain if AAV9 was directly injected into the SVZ, as laminin receptors are not exclusive to astrocytes (Jucker et al., 1993). However, direct injection of AAV9 into the cortex, striatum, thalamus, or hippocampus, shows little, if no transduction of astrocytes. Instead, parenchyma injections mostly transduce neurons (Cearley and Wolfe, 2006; Klein et al., 2008). This finding not only reiterates the importance of injection site choice but also the further characterization of AAV9 tropism. Interestingly, AAV9 also favors neuronal transduction if injected at a neonatal stage (Foust et al., 2009). The plausibility of using AAV9 to transduce SVZ astrocytes must still be investigated. One method to do this could be to characterize the laminin receptor distribution over the SVZ. If SVZ astrocytes show a high 37/67 kD laminin receptor expression, then AAV9 would serve as a good vehicle to induce transgene expression. 2.4. Comparison of AAV4, AAV5, and AAV9 AAV4, AAV5, and AAV9 all have benefits and drawbacks with regards to the transduction specificity of SVZ astrocytes. While AAV4 has already been shown capable of SVZ astrocytic transduction, it also heavily transduces ependymal cells, which might complicate the interpretation of results obtained with AAV4. AAV5 demonstrates more robust transgene expression compared with AAV4 and AAV9. It is also reported to use the PDGFR for host cell entry. As PDGFR is highly expressed in the SVZ, AAV5 may be a better candidate than AAV4 to specifically transduce SVZ astrocytes. However, no studies to date have shown that AAV5 can

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transduce astrocytes with high efficiency. Moreover, PDGFR is not exclusively expressed by astrocytes in the SVZ. Recent experiments evidence that AAV9 has one of the highest transduction specificity for adult astrocytes out of all known AAV serotypes. Yet the localization of its proposed receptor, 37/67 kD laminin receptor, has not been characterized in the SVZ. Therefore, the cellular transduction profile of AAV9 has to be further investigated within the SVZ. Recently, the Schaffer laboratory has carried out the direct evolution of AAV capsids through guided mutagenesis. Their experiments eventually produced two new AAV capsids that show higher in vivo astrocyte transduction rates than AAV2 in the rat striatum. These new capsids are namely L1-12 and ShH19 (Koerber et al., 2009). Though, it remains to be seen if these capsids are able to guide astrocyte-specific transduction more efficiently than AAV4, AAV5, and AAV9. AAVs can serve as safe and efficient vehicles for SVZ astrocyte gene transfer. AAVs are able to transduce both mitotic and postmitotic cells (Terzi and Zachariou, 2008) showing little to no host immune response (Feng et al., 2004). They can integrate into a host genome, albeit at a low efficiency (McCarty et al., 2004), allowing for long-term transgene expression (Zincarelli et al., 2008). Moreover, they are unable to replicate without the assistance of a helper-virus (Kaludov et al., 2001), attesting to their safety. In fact, AAVs are currently used in numerous clinical trials (Aucoin et al., 2008) and offer feasible avenues for gene delivery for various diseases like cystic fibrosis and Parkinson’s disease (Aitken et al., 2001; Kaplitt et al., 2007; Marks et al., 2008). To date, most clinical trials use such serotypes as AAV1 and AAV2. These serotypes have been used and so far proven safe for use in human therapeutics (Hajjar et al., 2008; Marks et al., 2008; Wang et al., 2009). Despite the lack of evidence, other AAV serotypes should presumably offer comparable biosafety to that of AAV1 and AAV2. AAVs have been reported to elicit a slight immune response in humans presumably due to previous natural exposure to wild type AAVs (VandenDriessche, 2009). The main effect of this immune response is to lower the efficacy of AAV transgene delivery and expression. Gray and Samulski hypothesize that the use of a less prevalent AAV capsid might lead to a lowered immune response (Gray and Samulski, 2008). More research is needed to determine the exact nature of the human immune response to each of the discussed AAV capsids. The existence of many AAV serotypes with different properties makes AAV a highly flexible system. Moreover, the insertion of specific promoters can both enhance targeting of transgene expression to specific cell types and may further boost transgene expression (Shevtsova et al., 2005). This viral vector system has two main limitations when it comes to transduction of SVZ astrocytes. First of all, the limited integration of the AAV vector genome into the host genome means that the transgene will be lost during mitosis in most cells. As SVZ astrocytes are quiescent dividing cells, transgene expression could be significantly reduced over time. However, AAVs display a higher degree of integration in quiescent hematopoietic stem cells. It is unknown whether this attribute will carry over to SVZ astrocytes. This ambiguity surrounding AAV integration should be taken in careful consideration when planning experiments. Importantly, a lack of integration can also be seen as an advantage, because the inserted transgene will not be passed onto the progeny of the SVZ astrocyte. Another drawback in the AAV system is its relatively small genome (4.7–5.2 kb). This small genome, in turn, restricts the size of an inserted transgene (Peel and Klein, 2000). Despite the size limitations of AAV, this system does offer an efficient means of double transduction (Reich et al., 2003 as cited in Shevtsova et al., 2005). These attributes combined with the ability of AAV4, AAV5, and AAV9 to proficiently transduce astrocytes show the efficacy of utilizing this system for targeted gene delivery to SVZ astrocytes.

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3. Lentiviral vectors Like AAVs, lentiviruses are efficient tools for in vivo gene transfer of both mitotic and post-mitotic cells (Azzouz et al., 2004). Lentiviruses are members of the Orthoretrovirinae subfamily of the Retroviridae family. They can be separated into two major classes, primate based viruses and non-primate based viruses. The primate group encompasses viral species such as human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). The nonprimate group contains viral species such as the equine infectious anemia virus (EIAV) and feline immunodeficiency virus (FIV; as reviewed in Azzouz et al., 2004). Lentiviruses are RNA containing viruses (Teschemacher et al., 2005), which are inherently more complex than the previously described AAVs. This complexity stems from their possession of regulatory genes and elements (Chang and Gay, 2001). Furthermore, the lentiviral capsid is encased by an envelope, which is encoded by its own set of genes (Stein et al., 2005). The additional regulatory sequences act as a double-edged sword. On one side, the capacity of these viruses is significantly larger (8–12 kb) than that of AAVs (Azzouz et al., 2004; Teschemacher et al., 2005) giving the freedom to insert more complex expression cassettes (Jakobsson and Lundberg, 2006). On the other side, however, these viruses can replicate on their own, without the presence of helper viruses, raising biosafety issues. Much work has been done to create a safe, replication deficient lentiviral system that is still capable of efficient in vivo gene delivery. Many of the viral auxiliary genes have been shown to be detrimental to cell survival. Therefore, minimal vector systems were created that lack all viral genes that do not play important roles in viral transduction (Chang and Gay, 2001). The HIV genome contains four such genes: nef, involved in the enhancement of viral infectivity, tat, an oncogene, vpr, associated with apoptosis and enhanced viral infectivity, and vpu, implicated in differentiation (Azzouz et al., 2004; Chang and Gay, 2001). After the removal of these four genes, the HIV genome is now comprised solely of long terminal repeats (LTRs), gag, pol, and rev. Furthermore, these precautions prevent the possibility of a wild-type HIV virus to be generated via recombination (Zufferey et al., 1998). Gag and pol are important lentiviral packaging elements, while rev plays a crucial role in the exportation of spliced or unspliced RNA into the host cell’s cytoplasm (Kim et al., 1998). The LTRs are essential for the synthesis and integration of DNA (Chang and Gay, 2001). Similar minimal vector systems have also been created for EIAV as well as FIV (Azzouz et al., 2004). The integration of a transgene into the DNA of the host cell allows for both its initial and long-term expression (Naldini et al., 1996). However, this integration may also lead to insertional mutagenesis (Meunier and Pohl, 2009) or other adverse effects, as lentiviruses typically insert a transgene into transcriptionally active genes (Mitchell et al., 2004). Problems stemming from integration can be circumvented by the use of integration deficient lentiviral vectors. Integration deficient lentiviral vectors show similar transgene longevity as AAVs, as their DNA remains in an episomal form within the cell nucleus (Philippe et al., 2006; YanezMunoz et al., 2006). That said, the prospect of an integrating lentivirus does allow for sustained transgene expression, ranging from several months to over one year (Azzouz et al., 2004; Zufferey et al., 1998). Integration also ensures the expression of a transgene in the progeny of the transduced cell. Another safety precaution is the packaging of a lentivirus into a different hetereologous envelope. This process, termed pseudotyping, is able to tailor tropism. Three types of envelopes, namely the vesicular stomatitis virus (VSV), the lymphocytic choriomeningitis virus (LCMV), and Mokola, hold promise for the transduction of SVZ astrocytes. The wildtype VSV is an oncolytic arthropod-borne virus (Lichty et al., 2004), while the LCMV is a

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rodent-borne arenavirus (Bonthius et al., 2002). Unlike VSV and LCMV, specifics of the wildtype Mokola virus remain, for the most part, obscure. It is a rabies-related virus, which is evidenced to be, like VSV, arthropod-borne (Ogunkoya et al., 1990). 3.1. VSV lentiviral envelope The VSV glycoprotein is the most commonly used envelope in in vivo research utilizing LVs, presumably due to its high stability and broad tropism (Mazarakis et al., 2001; Watson et al., 2002). It gains entry into cells via phosphatidylserine, an essential part of most cellsurface membranes (Lichty et al., 2004). Although the presence of phosphatidylserine is ubiquitous, some groups report that VSV displays a highly neuronal transduction preference (Mazarakis (Lai and Brady, 2002; Mazarakis et al., 2001)). Others attribute this reported neuronal preference to the use of unspecific, weak promoters and find that VSV is able to transduce all cells in the CNS with the same efficiency (Kang et al., 2002). Despite the inconsistent findings regarding astrocyte transduction, two independent groups report that a VSV pseudotyped HIV lentivirus vector is able to efficiently transduce murine SVZ astrocytes (Consiglio et al., 2004; Geraerts et al., 2006). In both studies, a reporter construct was inserted into a HIV-based lentiviral vector and encapsulated with a VSV envelope. Two weeks after SVZ parenchymal injection, reporter-positive cells were noted in the SVZ and the OB. Two, three, and six months later, the amount and distribution of the reporter-positive cells in the SVZ remained remarkably similar to that of the two week timepoint. Moreover, many cells along the RMS and in the granular layer of the OB also expressed the reporter construct. An ex vivo culture of the transduced SVZ cells showed that reporter-positive cells were able to form neurospheres that displayed stem cell like abilities to self renew over an extended period of time (Consiglio et al., 2004). Taken together, these results demonstrate the ability of the VSV pseudotyped HIV-based lentivirus to transduce SVZ astrocytes. However, this particular system also transduces neuroblasts as evidenced by the appearance of reporter-positive neuroblasts in both the RMS and OB in a short period after lentiviral injection. Delivery into the lateral ventricle resulted in little to no neuroblast transduction. Instead, this method resulted in the transduction of ependymal cells throughout the entire ventricular system along with tanycytes and some SVZ astrocytes. These findings illustrate the robust yet unspecific tropism of the VSV pseudotyped lentivirus. 3.2. LCMV lentiviral envelope LCMV pseudotyped lentiviruses show a more guided tropism than that of VSV. They gain entry into cells via a-dystroglycan, which plays an important role in linking cells to the basement membrane via extracellular matrix interactions. a-dystroglycan is found on both the postsynaptic terminals of neurons and the endfeet of astrocytes (Stein et al., 2005; Watson et al., 2002). Despite the presence of a-dystroglycan on both neurons and astrocytes, the wildtype LCMV shows a strong preference for astrocytes in the neonatal rat brain. In fact, Bonthius et al. (2002) report that neuronal transduction occurs after astrocyte infection and only in certain areas of the brain such as the cerebellum, dentate gyrus, OB, and periventricular area. This strong astrocyte transduction preference is carried over with all LCMV pseudotyped lentiviruses in both rats and mice (Miletic et al., 2004; Stein et al., 2005). A FIV-based, LCMV (WE54 strain) enveloped lentivirus displays a high transduction preference for both astrocytes and neural stem cells, when injected into the murine SVZ parenchyma (Stein et al., 2005). Though a LCMV pseudotyped lentivirus is able to robustly transduce SVZ astrocytes, it remains unclear whether it does so exclusively.

3.3. Mokola lentiviral envelope Unlike both VSV and LCMV, there is currently no known receptor for the Mokola virus. A Mokola pseudotyped vector is able to transduce murine neurons, oligodendrocytes, and astrocytes in vivo (Colin et al., 2009; Watson et al., 2002). Delivery of a HIVbased, Mokola pseudotyped lentivirus into the murine striatum resulted in the transduction of astrocytes (68%) and few neurons (9%). This observed astrocytic preference can be carried over to the SVZ. Alonso et al. (2008) injected a Mokola pseudotyped, HIVbased lentiviral vector into the SVZ of mice after cytosine arabinoside (Ara-C) treatment. Ara-C eradicates all fast-dividing cells, while leaving SVZ astrocytes intact (Alonso et al., 2008). The HIV-based, Mokola pseudotyped lentivirus was efficiently able to sustain transgene expression in these SVZ astrocytes over a prolonged period of time. Furthermore, these transduced SVZ astrocytes in both the SVZ and RMS were able to give rise to mature neurons in the OB. Ara-C treatment serves as an interesting option to ensure the exclusive transduction of SVZ astrocytes. Nevertheless, it remains unclear whether the Mokola envelope is able to direct restricted SVZ astrocyte transduction, or whether neuroblasts and/or transitory amplifying cells would also be transduced in a non-Ara-C treated brain. 3.4. Comparison of VSV, LCMV, and Moloka lentiviral envelopes VSV, LCMV, and Mokola pseudotyped lentiviral vectors all hold promise when targeting SVZ astrocytes. VSV is able to direct transgene expression over a broad host range (Mazarakis et al., 2001), while both LCMV and Mokola show a more preferential pattern of tropism, preferring astrocytes over any other CNS cell type. The LCMV and Mokola pseudotypes also offer a major advantage over VSV, as they are significantly less cytotoxic (Stein et al., 2005). This lower cytotoxicity not only allows for greater reliability in experimental results but also an easier transition towards human clinical applications. LCMV displays the lowest transduction efficiency of all the discussed pseudotypes. This aside, LCMV does seem to offer the most specificity in regards to neural stem cell and astrocyte transduction (Stein et al., 2005; Watson et al., 2002). Mokola also appears as a frontrunner in lentiviral pseudotype choice, as it displays the most efficient transduction rate with a seeming preference of astrocyte transduction. However, more research is needed in order to examine Mokola’s tropism in regards to other SVZ cell types, besides the SVZ astrocytes. The major advantage of using a lentivirus-based system, over AAV, is the large insert capacity. This large capacity allows for the insertion of a large transgene and/or reporter construct driven by a cell specific promoter. Astrocyte transduction efficiency could further be improved upon by pseudotyping with LCMV or Mokola. Lentiviral vectors serve as a safe and efficient method to induce transgene expression in SVZ astrocytes and their progeny. They do not elicit an extensive immune response that would cause severe side effects such as apoptosis or loss of transgene expression (Baekelandt et al., 2002; Meunier and Pohl, 2009). Furthermore, due to their ability to integrate into the host genome, lentiviruses offer excellent temporal transgene expression. This long-term gene expression along with their large carrying capacity makes lentiviruses an optimal candidate for SVZ astrocyte transduction. However, it must be kept in mind that even if exclusive SVZ astrocyte transduction can be achieved, a transgene will be continuously expressed in the transitory amplifying cells and neuroblasts stemming from the originally transduced astrocytes, as lentiviral transduction leads to a stable integration of the transgene. This integration may not be optimal in all experiments, but could prove crucial to others. In summary, the range of

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lentiviral pseudotypes in combination with the inherent flexibility of large expression cassettes make lentiviruses opportune vehicles for the delivery of transgenes to SVZ astrocytes. 4. Electroporation Electroporation (EP) is an efficient non-viral method of in vivo transduction. It utilizes a series of electric shocks made by passing current from an anode to a cathode to make small, transient pores in the cell membrane (Neumann et al., 1982). These pores act as transient gates that allow macromolecules to enter a cell. EP is an invaluable tool as it allows for the active insertion of a variety of macromolecules, including DNA, RNA, Anti-sense oligonucleotide (AON), and protein (Haas et al., 2001; Rols et al., 1998). Originally used for in vitro gene delivery (Neumann et al., 1982), EP has been adapted for successful in vivo use (Rols et al., 1998). In vivo EP makes use of low voltage, square wave pulses that have been shown to increase cell survival (Bloquel et al., 2004; Swartz et al., 2001). In vivo EP has many advantages including its low cost as well as the ability to transduce both post-mitotic and mitotic cells (Bigey et al., 2002; Bloquel et al., 2004). Furthermore unlike viral vectors, EP utilizes expression plasmids so it does not require elaborate production protocols and specialized facilities. Transgenes introduced into a cell via EP remain episomal. Therefore, EP is able to induce transgene expression from 36 h up to one month (Barnabe-Heider et al., 2008). The electrical current can be ‘‘aimed’’ by positioning the anode and cathode in order to achieve uniform, area-specific transduction (Bloquel et al., 2004; Gal et al., 2006). This ‘‘aiming’’ also theoretically allows for internal controls (Swartz et al., 2001)—so if the right SVZ was successfully targeted, the left SVZ could be used as a control. EP does have certain risks of toxic side effects. These side effects, namely cell death, are presumably due to extracellular media diffusing intracellularly during electroporation. Another cause of this toxicity results from less than optimal current delivery that may lead to big pores in the cell membrane, which are unable to reseal (Bigey et al., 2002; Bloquel et al., 2004). Despite these disadvantages, EP has been proven safe for use in the brains of experimental animals; as the electrical pulses do not lead to an inflammatory reaction, change in cell proliferation, seizures, or long-term abnormal EEG pattern. So far, only one study has used EP to directly target the adult SVZ. Plasmids were injected into the lateral ventricle and electroporated 1–2 min after needle retraction. EP was performed by holding electrodes to the adult mouse’s head and delivering five square wave pulses of 200 V, lasting for 50 ms each with 950 ms intervals. These parameters led to successful transduction of the SVZ, though the majority of transduced cells were ependymal cells, with only a small percentage (5.6%) of transduced GFAP-positive cells. Further, transgene expression was not restricted to the targeted SVZ, as transduced cells were also detected in the contralateral SVZ and the third ventricle (Barnabe-Heider et al., 2008). This observed aspecificity is most likely due to a combination of plasmid diffusion through the ventricle and lack of proper ‘‘aiming’’ in regards to electrode position. In contrast to intraventricular plasmid delivery, parenchyma injection shows a highly localized distribution pattern. BarnabeHeider et al. (2008) reported that plasmid injection into the adult hippocampus, followed by electroporation, led to transgene expression almost exclusively in the subgranular and granular layer. These results point out that parenchymal EP is extremely restricted in transduction area and aspecific in regards to cell type. This restricted localization is most likely due to a plasmid’s inability to deeply penetrate tissue, which shows the need for precise spatial targeting when injecting a plasmid. Aspecific cell population transduction can be overcome by using a cell specific promoter fragment regulating transgene expression.

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Moreover unlike other in vivo transduction techniques, EP is more suited to the transduction of larger plasmids. This quality makes the EP system more applicable for the insertion of a bulky promoter fragment to drive specific transgene expression. EP has the ability to transduce plasmids up to 52,500 bp (Kreiss et al., 1999). However, it must be kept in mind that increasing plasmid size decreases efficiency of transduction (see Table 3). The highest transduction efficiency is obtained with plasmids that are less that 10 kb. The charge of a given construct must also be taken into account when using EP. As current passes from anode to cathode during EP, more negatively charged macromolecules will yield a higher transduction efficiency (Slotkin et al., 2007). In summary, EP shows great promise for transducing SVZ astrocytes. It is a versatile method that allows for the manipulation of delivery parameters as well as desired macromolecules. It enables transient transgene expression from 36 h to 4 weeks (Barnabe-Heider et al., 2008). Further, EP is capable of the cotransduction of 2 plasmids with an efficiency of 90–99% and 3 plasmids with an efficiency of up to 98% (Barnabe-Heider et al., 2008; Swartz et al., 2001). These successful co-transduction rates enable the successful co-expression of a desired macromolecule along with a fluorescent reporter. In order to target adult SVZ astrocytes, a plasmid under the control of an astrocyte-specific promoter should be directly injected into the SVZ parenchyma and electroporated using the basic parameters presented by BarnabeHeider et al. (2008). The injection of a plasmid directly into the SVZ parenchyma, instead of the lateral ventricle, should result in a high transduction efficiency of SVZ astrocytes. This specificity would be achieved because the plasmid would not be able to diffuse into other brain regions as easily as seen with intraventricular injections. Furthermore, an astrocyte-specific promoter would prohibit unwanted transgene expression in ependymal cells. EP, under these proposed guidelines, would be a valuable approach in order to investigate transient transgene expression in SVZ astrocytes. 5. Promoter choice One of the issues in using viruses or EP to target SVZ astrocytes is the lack of inherent specificity. EP and viruses can be better aimed by the addition of an astrocyte-specific promoter. The next sections explore the efficacy of using the GFAP, aldehyde dehydrogenase 1 family member L1 (ALDH1L1), or glutamate aspartate transporter (GLAST) promoter to drive astrocyte-specific transduction (see Table 2). 5.1. The glial fibrillary acid protein promoters The use of the GFAP promoter to direct astrocytic specific transduction shows mixed results. Peel and Klein in 2000 used a 2.2 kb fragment of the GFAP promoter (gfa2; Brenner et al., 1994) to direct AAV transduction specifically to astrocytes in the spinal cord. The authors reported that most of the transduced cells were neurons. In fact, less than 30% of the transduced cells were astrocytes. However, other studies convincingly show that after parenchymal injection of an AAV2 or a baculovirus harboring a gfa2 promoter, that the gfa2 promoter is robustly able to guide astrocytespecific transduction (Feng et al., 2004; Wang and Wang, 2006). It must be noted that the gfa2 promoter has also been shown to transduce neurons in close proximity to astrocytes in brain regions such as the hippocampus, cerebellum, and cerebral cortex. This neuronal expression is variable and not always observed (Su et al., 2004). These data reiterate the importance of injection site choice as tropism can vary within different regions of the CNS. Though there are mixed findings regarding the gfa2 promoter, it seems that gfa2 may serve as a valuable method to direct SVZ

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26 Table 2 Astrocyte promoter specificity. Promoter

Length (bp)

Transduction pattern

Reference

ALDH1L1 gfa2 gfaABC1D gfa2(B)3 GLAST GLT-1

Unknown 2200 681 2569 636 773 or 2500

Probably astrocyte specific mostly astrocytes with variable neuronal expression astrocyte specific astrocyte specific, 10 higher than gfa2 astrocytes, radial glia, some neurons and oligodendrocytes astrocytes, ependymal cells, CA3 and CA4 neurons

Cahoy et al. (2008) Brenner et al. (1994) Lee et al. (2008) de Leeuw et al. (2006) Hagiwara et al. (1996), Regan et al. (2007) Liu et al. (2006), Regan et al. (2007), Romera et al. (2007), Su et al. (2003)

The use of an astrocyte-specific promoter can help specifically drive transgene expression in astrocytes. In addition to driving specific transgene expression, the use of such promoters lowers the host immune response when compared to a ubiquitously expressed promoter like Cytomegalovirus (CMV).

astrocytic specific transduction. The gfa2 promoter has been shown to direct astrocyte-specific transduction after parenchymal injection in AAV2 (Feng et al., 2004; Wang and Wang, 2006). It is interesting to note that the gfa2 promoter showed such success even though it was used with AAV2 (X. Feng, personal communication, July 2, 2009). AAV2 has a notorious neuronal tropism pattern (Tenenbaum et al., 2004), so the use of an astrocytespecific promoter in combination with a more ‘‘astrocyte-friendly’’ AAV has a high likelihood of limiting transgene expression to GFAP-positive cells. However, one of the major problems with using the standard gfa2 promoter is its size (2.2 kb). Size is an important aspect to consider when designing a plasmid, since it is restricted in viral technology and impacts transduction efficiency in non-viral approaches. To this end, it may not be practical to combine large transgenes or macromolecules with the bulky gfa2 promoter. The laboratory of Brenner conducted a study in 2008 that investigated which part of the gfa2 promoter was responsible for directing astrocyte specificity. They ultimately constructed a small (681 bp) promoter, termed gfaABC1D, which was found to exclusively transduce astrocytes in vivo. The gfaABC1D, was also able to increase transgene expression two-fold when compared to the original gfa2 promoter (Lee et al., 2008). Another variation on the gfa2 promoter is the gfa2(B)3 promoter. This promoter contains multiple enhancer elements, thereby increasing transgene expression 10-fold when compared with the gfa2 promoter. However, multiple enhancer elements mean a larger promoter (de Leeuw et al., 2006). Therefore, the gfa2(B)3 should only be considered when trying to achieve short-term, high transgene expression using a delivery system without strict size limitations. The gfaABC1D promoter fragment, on the other hand, may serve as a better option than both the traditional gfa2 promoter and the gfa2(B)3 promoter due to its compact size and high activity. 5.2. The aldehyde dehydrogenase 1 family member L1 (ALDH1L1) promoter GFAP promoters seem to be the most commonly utilized promoters to target astrocytes. But there are other astrocytespecific gene promoters that may be able to produce a broader and more robust astrocytic tropism, such as ALDH1L1. ALDH1L1 oxidizes formate and is thought to play an integral role in astrocytes’ metabolic processes (Neymeyer et al., 1997). A recent microarray study looking at the transcriptome of neurons, astrocytes, and oligodendrocytes of the mouse brain showed that ALDH1L1 is more heavily expressed in mature astrocytes than GFAP, as co-staining of ALDH1L1 and GFAP show that all observed ALDH1L1-positive cells are GFAP-positive and vice versa (Cahoy et al., 2008). ALDH1L1 is an especially interesting candidate to target SVZ astrocytes as it has been implicated in limiting proliferation of reactive astrocytes (Anthony and Heintz, 2007). More research is needed to determine the specificity of ALDH1L1 for SVZ cell types as there is a possibility that it may also target

ependymal cells (Neymeyer et al., 1997). Though no study to date has used or even characterized the ALDH1L1 promoter, ALDH1L1 still warrants investigation and could be an interesting alternative to the common GFAP promoters. 5.3. The glutamate aspartate transporter (GLAST) promoter Another interesting promoter candidate is GLAST; an important glutamate transporter required to maintain glutamate homeostasis in the brain. GLAST is expressed in GFAP-positive cells of the brain (Liu et al., 2006) as well as in GFAP-negative, vimentinnegative, and S100b-positive cells (Buffo et al., 2008), thus demonstrating its wide expression. GLAST is expressed by neurons, oligodendrocytes, radial glia, and astrocytes (Rothstein et al., 1994; Storck et al., 1992). Despite this broad tropism, the GLAST promoter has been successfully used to target mature astrocytes. Buffo et al. (2008) used the GLAST locus to drive inducible cre expression in 8–12 week old mice. They noted that GLAST was able to drive cre expression in half of the astrocytes of the neocortex. GLAST has also been successfully used in combination with in utero EP to target developing glia in the SVZ. This promoter was able to produce a 76–100% transduction rate (Gal et al., 2006). Although in utero targeting is not the best approach for transduction of mature astrocytes (Su et al., 2004), Gal et al. (2006) demonstrate that the GLAST promoter is able to induce robust transgene expression. GLAST can also be used to drive transgene expression in progenitor cells of the adult SVZ (Buffo et al., 2008; Mori et al., 2006). All of these spatial findings corroborate with GLAST’s temporal expression patterns. GLAST shows high developmental activity in the cortex, cerebellum, and SVZ. During adulthood, GLAST expression decreases in the cortex while it increases in both the SVZ and Bergmann glia of the cerebellum (Regan et al., 2007; Storck et al., 1992). Therefore, when using the GLAST promoter to drive transgene expression in the SVZ, the construct should be delivered locally into the SVZ parenchyma or lateral ventricle to avoid transducing unwanted brain regions. GLAST holds a major advantage to drive transgene expression in the SVZ as it is not expressed by ependymal cells (Liu et al., 2006). These findings along with the relatively small promoter size, approximately 636 bp (Hagiwara et al., 1996), make the GLAST promoter a suitable and realistic means to drive astrocyte and progenitor cell specific transgene expression in the adult SVZ. 5.4. Astrocyte-specific promoter comparisons Each of the promoters discussed above have their advantages and disadvantages. The gfa2 promoter is the most widely used promoter for astrocyte directed transgene expression. So data obtained with its use would be easily comparable to numerous other studies. However, its size poses major restrictions especially when driving large transgene expression. The same size limitations also apply for the gfa2(B)3 promoter. Therefore, the gfaABC1D, promoter would be the best choice out of all the GFAP promoters.

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The GLAST promoter, on the other hand, would allow for specific astrocyte and progenitor cell transgene expression in the adult SVZ without ependymal cell transduction. Notably, another glutamate transporter, glutamate transporter-1 (GLT-1), is also predominantly expressed by mature astrocytes (Desilva et al., 2007; Rothstein et al., 1994). Yet GLAST and GLT-1 rarely show colocalization (Regan et al., 2007). This lack of co-localization demonstrates that GLAST is only able to label a subset of all astrocytes. Though this would probably not pose a problem when targeting the SVZ, it should be taken into consideration if broader astrocyte-specific transgene expression is desired. ALDH1L1, out of all the astrocyte promoters, displays activity in the largest population of astrocytes. Therefore, ALDH1L1 may be able to drive transgene expression in a subset of astrocytes presenting limited proliferative capabilities. All of the promoters discussed above have the ability to specifically target SVZ astrocytes, as long as the construct is delivered locally into the SVZ. There is no one superior astrocyte promoter; rather promoter choice should be determined based upon experimental objectives. 6. Cre/LoxP technology The cre/loxP system is a versatile tool for conditional gene targeting. Conditional gene targeting refers the modification of a specific gene at a particular time-point and/or within a certain tissue/cell type (Schipani, 2002). The cre/loxP system is a binary system comprised of the cre enzyme that catalyzes conservative DNA recombination at predetermined loxP sites (Lewandoski, 2001). Temporal control can be achieved with the use of so called inducible cre lines. These mice contain cre fused with a ligand binding domain from an oestrogen or progesterone receptor (Lewandoski, 2001). For in vivo applications, loxP sites are usually inserted into intronic sequences utilizing site directed mutagenesis in embryonic stem cells (Camp et al., 2005; Nagy, 2000). The most applicable strategy for targeting astrocyte specific expression or down regulation would be to place loxP sequences in a cis arrangement flanking a target gene, as a whole, or around certain exons. The former case would result in the excision of the gene, while the latter would allow cre-mediated isoform expression. As these loxP sequences are small and come from a bacteriophage, they do not produce any disturbance in gene function (Nagy, 2000). Moreover, these sequences are highly unlikely to occur randomly in the eukaryotic genome (Schipani, 2002), thus ensuring proper targeting. Cell specific targeting is crucial to induce the recombination event in a select population of cells. As the loxP sequence has integrated into the genome, targeting relies on restricting cre expression. One successful strategy targeted astrocytes by fusing cre to the GFAP promoter. This so called 73.12 line expressed stable recombination mostly in astrocytes, with notably high recombination observed in the adult SVZ and SGZ. These astrocytes were observed to encompass both dividing and mature populations. However, there was also recombination in NeuN-positive neurons of the granular layer (Garcia et al., 2004). Another study attempted to target astrocytes by using the gfa2 promoter to drive cre expression. They crossed the gfa2-cre mice with a reporter line that expresses lacZ upon cre-mediated recombination. Though some astrocytes were successfully targeted, their results, as a whole, were disappointing. The double transgenic mice showed lacZ expression in a wide range of cells including mature neurons, ependymal cells, oligodendrocytes, astrocytes, and unidentified liver cells. This wide range of recombinated cells can be attributed to GFAP-positive multipotent neural stem cells that spawn diverse cell populations during development (Zhuo et al., 2001). It is interesting that the 73.12 line (Garcia et al., 2004) does not show the widespread recombination seen by Zhuo et al. (2001). Though the 73.12 line is mainly

27

astrocyte-specific, it is important to note that recombination was only analyzed in the adult mouse. Therefore, it is impossible to know the extent of embryonic recombination. Temporal aspects must also be controlled when specifically targeting mature astrocytes. Studies targeting astrocytes using inducible cre lines have met with mixed results. These studies have used a variety of promoters and gene loci to drive cre expression, including gfa2, GLAST, connexin 30 (Cx30), aquaporin 4 (Aqp4), and apolipoprotein E (ApoE). Of these, Aqp4 and ApoE mediated recombination have shown the least amount of success. Aqp4 heavily targets induced recombination in ependymal cells. ApoE mediated recombination is present at low levels ubiquitously throughout the brain; however, it is not restricted to the CNS, with the highest expression in the liver. The Cx30 promoter also seems like an unlikely candidate to specifically target SVZ astrocytes. Though it is able to drive astrocyte recombination, these astrocytes are primarily located in the mesencephalon and medulla. Furthermore, it causes substantial recombination in the skin (Slezak et al., 2007). Mori et al. (2006) achieved high SVZ recombination by inserting cre fused with the ligand binding domain for the estrogen receptor (CreERT2) into the GLAST locus. They induced cre expression by Tamoxifen injections. The SVZ showed a 65.9% efficiency rate, yet no details on specific SVZ cell types were reported. These GLAST::CreERT2 mice showed widespread astrocytic recombination in the striatum and cortex. However, there was small, yet significant, recombination also observed in NeuN-positive neurons and CC1-positive oligodendrocytes. A similar study using a GLAST transgene to drive CreERT2, showed that this construct was able to manipulate SVZ progenitor cells. However, like in the GLAST::CreERT2 line, this construct also induced widespread recombination throughout the brain (Slezak et al., 2007). The GCE mouse line has shown the most success in targeting SVZ astrocytes, of all inducible cre lines to date. This mouse harbors a gfa2 promoter driven CreERT2 construct induced by 4-hydroxytamoxifen. 79.5% of all recombinated cells in the SVZ were GFAPpositive with no observed recombination in neurons, oligodendrocytes, or ependymal cells. Most of the remaining recombinated SVZ population stained positive for the classic stem cell marker, LeX. Nonetheless, this pattern of astrocyte specificity was not exclusive to the SVZ, as high amounts of recombination were also seen in the cortex, hippocampus, mesencephalon, and cerebellum (Ganat et al., 2006). The gfa2 promoter has been shown to promote astrocyte-specific recombination in inducible cre lines. Using a similar construct, Chow, Zhang, and Baker (2008) induced cre recombination in 40–50 day old mice by Tamoxifen injections. Their results show that the gfa2 promoter drives astrocyte-specific reporter gene expression throughout the brain (Chow et al., 2008). But in contrast to Ganat et al.’ (2006) findings, less that 1% of all SVZ cells were targeted during recombination. This variation could be attributed to many factors including transgene integration loci, small variations in construct design, different synthetic steroids, or even age at time of induction. So even though, the GCE mouse seems to be the best cre line to induce astrocytic SVZ recombination, caution must be used as results were based solely upon cre activation at P5. Attention must also be drawn to the fact that there is no cre line that is able to restrict recombination solely to the SVZ. This broad tropism denotes a need for increased regional localization of induced cre recombination. Using viruses to deliver cre is one way to circumvent the regional aspecificity observed above. AAV or lentiviral vectors can be used to deliver cre into a specific brain region of a loxP line, thereby restricting cre-mediated recombination to the immediate area surrounding the injection site. This method is also significantly faster than the traditional cre lines, as there is no need for extra breeding. However, no study has yet shown a high

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recombination efficiency within the SVZ. Pilpel et al. (2009) injected a cre containing lentivirus in the lateral ventricles of neonatal mice. This resulted in recombination in the SVZ and a 20% recombination rate of migrating neuroblasts. However, specific quantification of recombinated cell types in the SVZ was not reported. Other studies show that cre containing AAVs are able to induce recombination in the adult brain (Ahmed et al., 2004; Berton et al., 2006). This viral mediated cre expression system may prove to be a good option when targeting SVZ astrocytes. Furthermore, cellular specificity could be achieved by driving the cre transgene cassette with an astrocyte-specific promoter. Another approach lies in the targeted delivery of synthetic steroids to inducible cre mice, like the GCE line. Synthetic steroids could be either injected by themselves, or be delivered directly into the SVZ parenchyma using a vehicle. No study has yet utilized this methodology, but one can hypothesize that this method would not only increase regional and cell type specificity but also increase recombination efficiency. The cre/loxP system is a reproducible and permanent manner of achieving conditional gene targeting in vivo. Inducible cre lines show recombination sensitivity in proportion to the amount of synthetic steroid delivered (Lewandoski, 2001). The viral mediated cre delivery systems also mimic this finding. However, a ceiling effect is often reported in inducible cre lines (Pilpel et al., 2009) and extreme levels of cre expression have been linked to DNA damage (Loonstra et al., 2001). The cre/loxP system is not able to induce recombination in 100% of targeted cells. SVZ recombination efficiencies have been reported from 0.1% to 79.5% (Chow et al., 2008; Ganat et al., 2006). This variation denotes the need for proper planning, pilot studies, and manipulation of variables to optimize recombination conditions. Once a given cre/loxP system is adequately optimized, it should yield specific recombination with relatively low background (Schipani, 2002). One huge advantage of this system is that once a mouse line has been established, it can essentially serve as a ‘‘renewable’’ source, thereby reducing innate variation among experiments. This advantage, however, comes at a cost. The creation of one cre or one loxP mouse takes a significant amount of time, and once generated, it still must be backcrossed and bred for at least 8 generations to achieve required stability (Casper and McCarthy, 2006). Though none of the afore mentioned cre systems has the ability to exclusively target SVZ astrocytes, there are many inbuilt variables, such as the ability to temporally regulate recombination, that can be played with in order to achieve a cre/loxP system that efficiently and exclusively target astrocytes in the SVZ. 7. Conclusion The subventricular zone is a dynamic niche with neurogenic properties that are as of yet not fully understood. SVZ astrocytes give rise to transitory amplifying progenitor cells, which in turn produce neuroblasts that under normal physiological conditions travel through the RMS, differentiate into neurons, and integrate into the OB. Under pathological conditions, such as ischemia, neuroblasts are found to migrate to the lesion site and differentiate into neurons, presumably in an attempt to repair the damaged network. Since neuroblasts originate from SVZ astrocytes, or more specifically B1 astrocytes, it is imperative to understand the basic nature of these astrocytes. The neurogenic properties of astrocytes can then be harnessed in order to rehabilitate the brain regions that are inflicted with a neurodegenerative disorder or a traumatic brain injury. What is the best method to specifically target these cells in vivo? All in all, no one method can be considered ‘‘the best’’ for targeting SVZ astrocytes. Instead, one must consider what method can be aptly utilized in order to tackle a specific question (see Table 3). AAV and LV delivery methods have the advantage of

transducing both post-mitotic and mitotic cells at a high efficiency (McCown et al., 1996; Peel and Klein, 2000). AAVs show longlasting transgene expression, for up to one year or longer (Belur et al., 2008; Haberman et al., 1998). Nevertheless, they have a small cloning capacity (4.7–5.2 kb) along with a low integration rate (0.1–15%; McCarty et al., 2004; Wu et al., 2009). Notably, AAVs may possibly integrate more efficiently into quiescent SVZ astrocytes. However, the exact integration properties in regards to SVZ astrocytes remain to be seen. LVs, on the other hand, have a significantly higher cloning capacity (8–12 kb) and stably integrate into the host genome (Azzouz et al., 2004; Teschemacher et al., 2005). Using EP to deliver a construct to a cell results in transgene expression for up to one month, at which point in time the construct is degraded. Despite its short expression time, EP is quicker and more cost effective than both viral delivery systems and cre/loxP systems. The cre/loxP system offers a permanent change in the host genome. Though, it must be noted that mosaic recombination is often observed in inducible cre systems (Johnson et al., 2009). A benefit of this system is that a cre/loxP mouse essentially serves as a renewable resource. However, the time, effort, and cost it takes to produce a stable mouse line, make the cre/loxP system unrealistic for preliminary and exploratory experiments. Integration of a transgene into the host cell is an important consideration when planning SVZ experiments. If a construct is integrated, and driven by a general promoter instead of an astrocyte-specific one, then the progeny of the SVZ astrocyte will also express the inserted transgene. On the other hand, an episomal transgene, or an integrated transgene driven by an efficient astrocyte-specific promoter will only be expressed by astrocytes. Integration allows for sustained transgene expression, especially in an environment where SVZ astrocytes undergo mitosis. Mitosis will cause an episomal transgene to gradually dilute and eventually disappear. Confounding results may be obtained if one transduction method results in both integration and episomal forms of the inserted transgene. These integration properties may help in the selection of the desired delivery system. One of the major difficulties when transducing SVZ astrocytes is avoiding ependymal cell transduction. There are two main strategies that can be used in combination with both viral vectors and EP. Firstly, constructs/viruses should be injected directly into the SVZ parenchyma, not into the lateral ventricles. Injection into the fluid filled lateral ventricles allows the construct to quickly diffuse away from the targeted site. Moreover, ependymal cells essentially block the entry of a delivered construct into the SVZ as they are more readily transduced due to their immediate proximity to the injection site (Geraerts et al., 2006). Secondly, cell specific promoters can be used to target a desired population of cells. Another benefit of utilizing a cell specific promoter in conjunction with a viral vector, such as LV, is that the host displays a lowered immune response and is more efficiently able to translate the inserted transgene into a protein (Follenzi et al., 2007). Injection of a construct driven by an astrocytic promoter into the SVZ parenchyma should result in the exclusive transduction of SVZ astrocytes. There are many different delivery vehicles capable of inducing transgene expression and modulating endogenous gene expression in SVZ astrocytes in vivo. As discussed above, each of these systems has its individual advantages and disadvantages (summarized in Table 3). All of these variables should be taken into account when planning an experiment revolving around the transgene modulation in SVZ astrocytes. It is imperative to carefully consider methodology choice in relation to specific experimental questions, as every question requires its own approach. There is no supreme method that specifically and efficiently targets SVZ astrocytes in vivo; as each of the discussed delivery systems requires further

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Table 3 Summary of in vivo transduction methods for targeting SVZ astrocytes. Delivery vehicle

CNS cell type

Integration

Expression time

Main advantages

Main disadvantages

AAV4

Mitotic and post-mitotic cells; astrocytes and ependymal cells1,2,3

Sometimes, 0.1–15%4

Long-term5

- Small transgene size (4.7–5.2 kb) carrying capacity

AAV5

Mitotic and post-mitotic cells; all CNS cell types1,6,7,8,3 Mitotic and post-mitotic cells; astrocytes and neurons9,3 All10

- Novel modifications of various serotypes may result in vectors with unexpected properties beneficial for transduction of SVZ astrocytes18. So flexibility of AAV holds great promise - Easy transition to clinical applications

Yes, Stable10

Long-term10

- Established lines allow for generations of experiments with innately small background variation

Cre/LoxP (inducible)

All11

Yes, Stable11

Long-term, after induction11

- Lifetime expression of conditional gene modification - Use in combination with a viral vector encoding Cre may offer specific benefits, e.g. local targeted gene knock-out

- Inducible Cre/LoxP lines show ceiling effects

Electroporation

All12

No

Short-term; 36 h–1 month13

- No need for specialized production facilities - Able to transduce using a variety of macromolecules

- Short expression time - Lowest transduction efficiency of all discussed methods

Lentivirus

Mitotic and post-mitotic cells; neurons and glia14

Yes, Stable15,16

Long-term15,16

- Stable integration - Large (8–12 kb) carrying capacity

- Transgene will be expressed in the targeted astrocyte along with its progeny. However this can be both an advantage and disadvantage, depending on the condition or needs of the experiment - Special production facilities are required to generate viral vectors

AAV9

Cre/LoxP

- Uncertainty about its genomic integration in SVZ cells - Special production facilities are required to generate viral vectors - Establishing a new Cre/LoxP mouse requires much time

Each delivery vehicle offers its own unique transduction preferences and expression times. Most of the delivery vehicles discussed in this paper offer long-term expression in both mitotic and post-mitotic cells. However, each vehicle differs in its tropism and ability to integrate. These variables must be taken into consideration when tailoring methodology. All of the discussed methods hold promise to specifically and efficiently target SVZ astrocytes. The choice of which method to put in play will heavily rely on experimental questions as well as available resources. References: 1. Davidson et al. (2000); 2. Liu et al. (2005); 3. Terzi and Zachariou (2008); 4. McCarty et al. (2004); 5. Zincarelli et al. (2008); 6. Di Pasquale et al. (2003); 7. Sevin et al. (2006); 8. Watson et al. (2005); 9. Foust et al. (2009); 10. Nagy (2000); 11. Lewandoski (2001); 12. Bigey et al. (2002); 13. Barnabe-Heider et al. (2008); 14. Jakobsson and Lundberg (2006); 15. Geraerts et al. (2006); 16. Oehmig et al. (2004); I7. Hermens and Verhaagen (1998); 18. Koerber et al. (2009).

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