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Kittler JT, Moss SJ, editors. The Dynamic Synapse: Molecular Methods in Ionotropic Receptor Biology. Boca Raton (FL): CRC Press/Taylor & Francis; 2006.

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The Dynamic Synapse: Molecular Methods in Ionotropic Receptor Biology.

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Chapter 13Lentivirus-Based Genetic Manipulations in Neurons In Vivo

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13.1. INTRODUCTION

Various classes of retroviruses, adenoviruses and adeno-associated viruses have been successfully adapted for development of recombinant vectors with the aim of long-term gene delivery to different cell types in different tissues [1]. Lentiviruses belong to a class of retroviruses that efficiently infect both dividing and non-dividing (post-mitotic) cells, making the recombinant lentiviral vectors applicable for stable, long-term gene delivery to neurons [1–4]. The latest, state-of-the-art generations of the lentiviral vectors have a large (about 9 kilobases) transfer capacity, and gene delivery via these vectors is devoid of cellular cytotoxicity or humoral response. Thus, whereas these vectors are being developed primarily for clinical applications in gene therapy, they provide an excellent and easy-to-use tool for gene manipulation in cultured neurons as well as in neurons in vivo, something that has been long missing in basic neuroscience research.

Here, we first briefly review the history of the development of the currently used lentiviral vector systems, and then focus on the use of these vectors for gene delivery and gene knockdown in pyramidal neurons in rodent brains. We discuss the experimental advantages of stereotactic injections of lentiviral particles, including the high spatiotemporal control over the introduced genetic manipulation and the fact that only a small population of neurons is affected within otherwise intact neuronal networks. In our view, lentiviral vectors provide in many ways an optimal tool for the study of gene functions in small populations or even individual neurons in vivo, which can be combined with physiological analysis of the infected neurons either in vivo or in vitro.

13.2. LENTIVIRUS-BASED VECTOR SYSTEMS

The first efficient lentiviral expression system was derived from the human immunodeficiency virus type 1 (HIV-1) and consisted of the following three vectors: a packaging vector expressing the structural Gag and Gag-Pol proteins as well as regulatory and most of the viral accessory proteins; an envelope vector expressing heterologous surface glycoproteins — either amphotropic envelope of murine leukemia virus or G glycoproteins of vesicular stomatitis virus (VSV-G); and a transfer vector containing human cytomegalovirus (CMV) promoter for heterologous protein expression [5]. The biosafety of the system was greatly improved in the next vector generations, first by deletion of all accessory genes from the packaging vector [6] and second, by the development of the so-called self-inactivating (SIN) transfer vector, which lacks the transcriptional activation sites from the 3′ LTR U3 region (Figure 13.1) [7,8]. The inactivation of the long-terminal repeat (LTR) transcriptional capacity is particularly important with respect to reducing the risk of vector mobilization and recombination with latent retroviral sequences in the host cell genome.

FIGURE 13.1. Latest generation of the lentiviral self-inactivating (SIN) vector.

FIGURE 13.1

Latest generation of the lentiviral self-inactivating (SIN) vector. Schematic representation of the SIN lentiviral vector (top) and its integrated proviral form (bottom). The cis-acting elements of the vector are indicated as white boxes. In producer (more...)

Next to the HIV-1-based expression systems, other primate as well as non-primate lentivirus-based vectors have recently been developed [1,9]. Of these, non-primate vectors derived from feline immunodeficiency virus (FIV) [10] and equine infectious anemia virus (EIAV) [11] are the furthest developed, and were both shown to efficiently infect neurons in vitro and in vivo. Whereas these vectors offering any advantage in terms of biosafety in comparison to HIV-1-based vectors is not clear, the EIAV vectors appear to be more efficient for gene delivery via axonal retrograde transport when pseudotyped with rabies glycoproteins [12]. With respect to transfer capacity and stability of expression, the latest FIV and EIAV vectors appear to be comparable to those derived from HIV-1 [13,14].

13.3. LENTIVIRUS-BASED HETEROLOGOUS EXPRESSION

Recombinant lentiviruses are typically produced (“pseudotyped”) with the VSV-G glycoproteins, mainly because it allows easy concentration to high titers by ultracentrifugation [15]. At the same time, because the VSV-G glycoproteins bind ubiquitous phospholipid components of the plasma membrane rather then a specific cell surface receptor, such viruses have an extremely broad host-cell range. The strength as well as cell-specificity of heterologous expression from the VSV-G-pseudotyped lentiviral vectors is thus determined by selection of a specific recombinant polymerase II promoter that drives the expression of the gene of interest. We have recently tested several promoters for expression in rat cortical pyramidal neurons in vivo [16]. This work showed that Synapsin I promoter was the most efficient during the second postnatal week, whereas calcium/calmodulin-dependent protein kinase II (-CaMKII) promoter was the strongest from the third postnatal week on. Within 1 week from infection, the level of expression of enhanced green fluorescent protein (EGFP) was sufficient for in vivo two-photon imaging at the resolution of dendritic spines and axonal branches [16]. Both promoters drove EGFP expression selectively in neurons versus glia, and the α-CaMKII promoter was selective for pyramidal neurons versus interneurons (some misexpression was observed, and is to be expected, due to integration of the viral vector backbone to gene-rich transcriptionally active regions) [17]. The expression in cortical neurons was stable for at least 2 months (the longest period tested), and it may be expected to stay stable throughout the life of the animal [18]. Finally, the intrinsic properties of the infected neurons, as analyzed by electrophysiology in vitro in slices and in vivo in anesthetized animals, were not different from uninfected control cells [16]. Thus, the vectors are well-suited for high-resolution imaging of structural synaptogenesis of cortical neurons in young as well as adult animals. Furthermore, combination of EGFP expression with expression of a second gene or downregulation of endogenous genes via RNA interference (see below) makes possible the use of such vectors for studying gene functions in synaptogenesis in vivo.

13.4. LENTIVIRUS-BASED GENETIC MANIPULATIONS

In research applications, to be able to co-express two proteins — a protein function of which one wishes to study and a fluorescent protein (typically EGFP) to label the infected cells — is often important. An easy approach is to use a bicistronic vector, where the second protein is expressed downstream of a viral internal ribosome entry site (IRES) (Figure 13.2a) [19]. However, the IRES-based initiation of translation tends to be considerably less efficient, resulting in an unequal expression of the two proteins. Combination of two promoters, either in tandem [20] or in a reverse orientation separated with a “stuffer” sequence [21], was described as an alternative approach for expression of two proteins from lentiviral vectors. However, we observed strong transcriptional interference between our Synapsin I and α-CaMKII recombinant promoters when adapted, in either orientation, into the self-inactivating lentiviral vector [22]. Recently, an elegant solution to this problem was published by the laboratory of Luigi Naldini, which showed that a synthetic bidirectional promoter composed of a minimal core of the CMV promoter (in one direction) and a cellular promoter (ubiquitin or phosphoglycerate kinase promoter in the opposite direction) drive expression of two proteins at a strength and cell-specificity determined by the cellular promoter [23]. We are presently testing this system for the α-CaMKII promoter (Figure 13.2b).

FIGURE 13.2. Lentiviral vectors for genetic modification in neurons.

FIGURE 13.2

Lentiviral vectors for genetic modification in neurons. (a) Vector with 1.3-kb promoter fragment of α-CaMKII drives strong expression in pyramidal neurons in cortex and hippocampus. Insertion of IRES site makes for bicistronic vector for co-expression (more...)

Gene downregulation (knockdown) via RNA interference (RNAi) has become an extremely popular approach to achieve a loss-of-function manipulation in many different cell types [24,25]. Briefly, introduction of double-stranded short-interfering RNAs (siRNAs, typically 21 base pairs) into a cell causes an activation of a multi-protein complex termed RISC (RNA-induced silencing complex), and a subsequent degradation of cellular mRNAs containing a homologous region to the siRNA sequence. Recently, expression of siRNAs in mammalian cells was achieved from plasmids containing a polymerase III promoter, e.g., the small nuclear RNA U6 promoter [26]. In this approach, siRNA sequence is expressed as a fold-back short hairpin RNA (shRNA) that is post-transcriptionally processed into typical siRNA via cellular RNase Dicer. We as well as others [27,28] have established lentiviral vectors containing one or two polymerase III promoters for shRNA expression together with a polymerase II promoter for expression of a fluorescent protein (Figure 13.2c) [16]. Thus, next to the option of heterologous expression of one or two genes (above), achieving a knockdown of endogenous genes in neurons is now possible — again in a combination with expression of a fluorescent protein to label the infected cells.

13.5. DELIVERY OF LENTIVIRAL PARTICLES INTO THE BRAIN

Lentiviral vectors were shown to efficiently infect neurons in vivo in rodent as well as primate brains in the nigrostriatal system and hippocampus [5,6,29–34]. An important aspect to consider with respect to lentivirus delivery to the brain parenchyma is the extent of diffusion of the viral particles in the brain extracellular space (ECS). The ECS between cells in the vertebrate central nervous system is estimated to be between 20 to 40 nm [35,36]. In contrast, HIV-1 particles are quite large —approximately 80 to 100 nm in diameter (database of the International Committee on Taxonomy of Viruses, www.ncbi.nlm.nih.gov/ICTVdb) — and the particle size thus should strongly limit their free diffusion in the brain (note that the VSV-G pseudotyped and wild-type HIV-1 particles seem to be of the same size) [37]. From our experience, injections of different volumes (ranging from about 25 to 200 nl) of lentiviruses into the rat cortex or hippocampus (animals greater than P8) result in infection within a spherical region of a similar size, approximately 400 to 600 μm diameter (Figure 13.3a). This result supports the notion that the viruses encounter substantial hindrance in the ECS and can achieve only a limited spread. (We have not tried larger injections, as these would likely cause some damage in the cell-dense cortical or hippocampal regions.)

FIGURE 13.3. Infection of neurons in vivo.

FIGURE 13.3

Infection of neurons in vivo. (a) Examples of sparse and dense infections in the barrel cortex. Injection of a small volume (about 25 nl, first panel) or a large volume (about 200 nl, second panel) of the same viral stock results in a correspondingly (more...)

Because the VSV-G coat binds directly to membrane phospholipids [15], the particles can be endocytosed not only into cell somata but also into dendrites and axons within the injection area. From our experience with injections in the rodent barrel cortex, high-rate infection in the superficial cortical layers (layer 2/3) is typically accompanied by infection of few (10 to 20) deep layer-5 pyramidal neurons that send their apical dendrites through layer 2/3 all the way to the layer 1 (Figure 13.3c). This behavior suggests that dendritic uptake of the virus and its subsequent transport to the soma is possible but rather inefficient. At the same time, finding any infected neurons farther away from the injection site is extremely rare, indicating that axonal uptake or retrograde transport almost never occur. Interestingly, even when pseudotyped with rabies glycoproteins, HIV-1 vector-based particles are very inefficient in retrograde infection [37,38]. In contrast, rabies-pseudotyped EIAV vectors appear to undergo efficient retrograde transport [12]. Presently, no explanation exists for this inconsistency.

13.6. EXPERIMENTAL APPLICATIONS

Due to the limited spread of lentiviruses in the vertebrate ECS, injection-based delivery of the lentiviral vectors is not practical for gene manipulations in large populations of neurons — for example, in an extensive cortical region or large portion of the hippocampus. On the other hand, stereotaxic injections of recombinant len-tiviral particles provide a high spatiotemporal control over the genetic manipulations, making the targeting of small neuronal populations with distinct cellular functions easy, such as a subpopulation of pyramidal neurons in the CA1 hippocampal region or in the cortex (Figure 13.3b and Figure 13.3c), at a defined time in the postnatal development. In addition, the fact that only a small number of neurons is infected means that gene functions can be altered in a way that would result in a lethal phenotype or in an activation of compensatory mechanisms if the entire brain or whole brain subregions were altered. This might allow the study of in vivo functions of genes that otherwise would be possible to study only using neuronal cultures.

The types of experiments that can be easily carried out with the lentiviral vectors include, for example, studies of protein trafficking when wild-type and mutant forms of GFP-tagged proteins are expressed in vivo and analyzed for subcellular distribution “post-hoc” from fixed-brain sections, or studies of cellular physiology when gene functions are altered via viral infections in vivo and analyzed in vitro for changes in synaptic transmission or synaptic plasticity in acutely prepared brain slices. We have recently shown that the Synapsin I and α-CaMKII promoter-based vectors can also be used for in vivo two-photon time-lapse imaging of morphological dynamics of dendritic spines and axonal projections of infected EGFP-expressing cortical neurons [16]. Similarly, imaging of EGFP-tagged proteins could be used for time-lapse imaging of trafficking of synaptic proteins in vivo, an application that is yet to be explored. Finally, perhaps the most exciting use of the lentiviral vectors for studying gene and cellular functions in neurons could be in combination with in vivo two-photon targeted patching (TPTP) [16,39]. We have recently shown a “proof of principle” for TPTP from lentivirus infected cells by recording sensory-evoked responses and receptive field maps from EGFP-expressing layer 2/3 pyramidal neurons in the somatosensory barrel cortex in anesthetized animals [16,40]. The method of in vivo targeted whole-cell recording from genetically altered cortical neurons can thus be applied to study gene and cellular functions in individual neurons in the intact cortex, either during early postnatal development or in the adult in a cortical region of choice.

13.7. CONCLUSIONS

In summary, the approach of lentivirus-based gene manipulations in neurons in vivo, as described here, offers a number of experimental advantages for cellular neuroscience, including the ease of viral production [29], the large transfer capacity of the vector and the lack of any cellular or humoral response associated with injection of the virus into the brain. Stereotaxic delivery of lentiviruses to specific brain regions provides an alternative to traditional mouse genetics with high spatiotemporal control over the genetic manipulation and a short time from experimental design to data collection and analysis.

13.8 ACKNOWLEDGMENTS

We thank Peter H. Seeburg for his generous support. Dr. Dittgen is supported by GIF grant (I-733-60.13/2002) to Pavel Osten. We thank Damian J. Haydon-Wallace for comments on the manuscript.

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