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The central nervous system (CNS) orchestrates diverse and complex biological processes by clusters of neurons wired through synaptic connections and the glial interface. The goal of brain research is to understand both the structure and the function of these communications. One of the main obstacles when studying neuronal circuits is the complexity of the organization of the CNS itself. The hypothalamus is a key player in the regulation of body energy homeostasis (Myers and Olson, 2012;Waterson and Horvath, 2015;Timper and Br€ uning, 2017). Its complexity is due to the high level of neuronal interconnectivity and the fact that regulatory elements and mechanisms for specific expression may differ between similar cell types. This can be the case even between parvo-and magnocellular variants of the same cell type located in the same area, such as in the paraventricular nucleus (PVN) (Ferguson et al., 2008). The molecular characterization of different hypothalamic neuronal populations has ultimately allowed for the generation of transgenic mice for the study of many genes involved in the regulation of energy homeostasis. For this purpose, two main strategies have been developed: d A modified target gene can be introduced into mouse embryonic stem cells (mESCs) (Court et al., 2002;Sun et al., 2003). The inoculation of mESCs carrying the genetic modification into blastocysts and their implantation into foster mothers will eventually generate a chimeric mouse able to transmit the mutant gene to their progeny (Okita et al., 2007). Further crossing of the chimeric mouse with wild-type mice allows for the isolation of the homozygous gene-targeted mouse. d The Cre-lox system is used as a genetic tool to control sitespecific recombination events in genomic DNA (Gu et al., 1993). The system is based on the ability of the lambda phage-derived Cre protein to recognize specific sites called loxP. LoxP sites are formed by two series of 13 bp palindromic sequences divided by 8 non-symmetrical bp.
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The Cre protein is able to mediate the recombination of the DNA sequence flanked by two loxP sites (Gu et al., 1993). By using a tissue-and cell-specific promoter, it is possible to generate a mouse line in which the Cre protein expression is spatially confined to that area in which the selected promoter is active. Crossing a Cre-specific mouse line with one carrying the target gene flanked by two loxP sites will ultimately result in a spatially restricted gene editing.
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Nevertheless, these approaches have limitations. For example, it is not always possible to target a specific gene in the mESC to obtain a living mouse because the physiological expression of the target gene itself could be essential for normal embryogenesis and development. Further, genetic manipulation occurring during early developmental stages might induce compensatory effects and, ultimately, bias the study outcome. Even if these issues can be overcome by using the Cre-loxP gene-editing strategy, this still would not allow for the confinement of the gene editing to specific neuronal circuits within a restricted volume. To surmount these difficulties, different viral vectors have been developed over time to deliver genetically encoded tools for the study of neuronal populations within neural circuits (Arenkiel andEhlers, 2009: Zagoraiou et al., 2009). Several viral vectors have been tested in vivo in the CNS, including lentiviral (LV), adeno-associated viral (AAV), adenoviral (AdV), and herpes simplex viral (HSV) vectors (Terzi and Zachariou, 2008;Lundberg et al., 2008;Segura et al., 2008;Berges et al., 2007). Such tools can be used successfully for a wide range of genome editing (such as generation of knockout, conditional knockout, and knockin lines and expression of light-sensitive ion channels for optogenetics studies). Choosing the appropriate viral vector is of extreme importance because, in most cases, their utilization is not interchangeable, and it often represents the key step toward the success or failure of the study itself. We will briefly describe some of the approaches for which viral vector technologies have been used and then focus on LV and AAV vectors and their use in the study of the hypothalamic circuits involved in the regulation of energy homeostasis.
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Recombinant Virus-Based Technology Versatility: From the Lab to the Clinic When aiming to modify the genetic makeup of cells in a targeted and precise manner by introducing genetically encoded tools, it is no wonder that the use of viruses has led the field. The technology has evolved to efficiently deliver genes, in the form of either DNA or RNA, into mammalian cells. Indeed, this characteristic has been the central element in the development of multiple approaches for which viral vectors are being used. In a very elegant study, Fields et al. (2012) used AAV vectors to elucidate the genetic elements responsible for the cell-type-specific expression of oxytocin in a specific subtype of neurons within the magnocellular neurons (MCNs) of the rodent supraoptic nucleus (SON). Recombinant AAV (rAAV) vectors carrying different oxytocin gene promoter deletion constructs, together with the reporter gene enhanced green fluorescent protein (EGFP), were generated and delivered into the MCNs. Using this method, co-localization studies of oxytocin/EGFP expression allowed the identification of regulatory regions in the oxytocin promoter that confer the specific neuropeptide phenotype. Notably, viral vectors represent the most popular means in the optogenetic field, i.e., the technology of genetic intervention based on expressing light-activated ion pumps into cells to control/modulate their activity (Knobloch et al., 2014). Boyden et al. (2005) first demonstrated the initiation of a light-dependent action potential in mammalian neurons via viral delivery of an opsin protein, channel-rhodopsin 2 (Boyden et al., 2005). In this field, viral vectors are particularly useful due to the rapidity of their experimental implementation and their ability for temporally and spatially defined intervention (Knobloch et al., 2014). The specificity of certain viruses to selectively transduce neurons has been used in tracing studies of neuronal circuits to analyze neuronal connectivity at high resolution (Luo et al., 2008;Callaway, 2008). Herpes viruses and rabies virus are used as viral vectors carrying genetically encoded tracing systems, allowing one to trace connections to or from particular cell types (Sauer et al., 1987;Schnell et al., 1994;Mebatsion et al., 1996;Callaway 2008).
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On the clinical side, the use of viral vector technology has emerged as a significant option for the treatment of several disorders. In monogenetic hereditary diseases, viral vectors have been used to achieve gene replacement by providing a normal copy of the defective gene. An example in this context is the currently ongoing phase I clinical trial for spinal muscular atrophy (SMA) (https://clinicaltrials.gov/ct2/show/record/NCT02122952), a fatal condition affecting the motor neurons, resulting from the deletion or mutation of the survival motor neuron-1 (SMN1) gene along with retention of the SMN2 gene. In this clinical trial, gene replacement is accomplished by using SMN1-expressing AAV9 vectors (Bevan et al., 2010(Bevan et al., , 2011;;Foust et al., 2010;Mendell et al., 2017). Gene transfer by rAAV vectors has been successfully used to study the involvement of different hypothalamic neuronal pathways in regulating energy homeostasis. In this context, by using rAAV vectors encoding leptin, an adipokine produced in proportion to fat mass, several studies have elucidated its role in decreasing the activity of neurons expressing the appetite-stimulating NPY factor while increasing the activity of the appetite-suppressing melanocortin signaling (Dhillon et al., 2001). In a very elegant study, Cao et al. (2009) used rAAV to focally overexpress the brain-derived neurotrophic factor (BDNF) in the hypothalamus of mice and developed a rAAV vector-based autoregulatory system to control therapeutic gene expression mimicking the body's natural feedback regulation of BDNF (Cao et al., 2009). Specifically, the autoregulatory capacity was achieved by designing a rAAV vector containing two expression cassettes, one to express BDNF under a constitutive promoter and the other to express a microRNA targeting the same transgene driven by a promoter responsive to BDNFinduced physiological changes (Cao et al., 2009).
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Recently, important progress has been made in the field of genome editing with the discovery of the CRISPR-Cas9 tool (Barrangou et al., 2007;Mali et al., 2013;Cong et al., 2013;Hsu et al., 2014). It utilizes a complex of a nuclease called Cas9 and a synthetic guide RNA (sgRNA) that recognizes target DNA by Watson-Crick base-pairing (Mali et al., 2013;Cong et al., 2013). Guided by sgRNA, Cas9 targets the protospacer adjacent motif (PAM) and generates site-specific double-strand breaks. These double-strand breaks are subsequently repaired either by non-homologous end joining (NHEJ) or by homology-directed repair (HDR) upon the existence of a donor template. NHEJ is more efficient than HDR but is error prone and may produce an indel (insertion or deletion) mutation, whereas HDR can provide a precise gene modification (Bibikova et al., 2003). Ex vivo and in vivo delivery of the single components of CRISPR-Cas9 by viral vectors have revolutionized the field of gene therapy. The possibility of combining the versatility of the CRISPR-Cas9 technology and the high specificity in targeting different tissues and cells is currently being explored for potential therapeutic applications in different human pathologies (Kennedy et al., 2015;Yin et al., 2014;Schwank et al., 2013).
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LVs are closely related to retroviruses, both belonging to the family Retroviridae. Their genome consists of a dimer of the linear positive-sense single-stranded RNA. However, LVs can be considered as advanced or more complex retroviruses due to the presence in their genome of several regulatory genes beyond canonical common retroviral genes. The major host cells of LVs are cells of monocyte/macrophage lineage and CD4 + lymphocytes (Gendelman et al., 1985(Gendelman et al., , 1986(Gendelman et al., , 1990)). LVs have similar genetic composition, with the structural genes gag, pol, and env encoding the core proteins reverse transcriptase and envelope glycoproteins, respectively. The viral life cycle of a LV starts with the interaction of the virion with the target cell through the viral surface glycoprotein with the main cellular receptor represented by the CD4 molecule of the T lymphocytes and monocytes/macrophages. The next step consists of partial uncoating of the virion and the generation of a double-stranded DNA copy of the viral RNA genome by the virion-associated reverse transcriptase. The viral DNA is then integrated into the host genome, generating a latent infection. LVs have long been used as tools to affect gene expression. The most effective LV vectors are those derived from the human immunodeficiency virus (HIV). Upon appropriate modifications, LVs are able to infect quiescent cells and have been shown to transduce most of the cell types within the CNS both in vitro and in vivo, including pre-mitotic and post-mitotic neurons (Blo ¨mer et al., 1997;Howarth et al., 2010), neuronal stem cells (Consiglio et al., 2004), and astrocytes and oligodendrocytes (Jakobsson et al., 2004;Miletic et al., 2004;Naldini et al., 1996), thus making them an attractive tool for the study of the CNS.
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The development of strategies to insert heterologous proteins into the virus envelope has led to the formation of so-called pseudotyped LV vectors. These approaches have further expanded the host range. Another attractive aspect of LVs as vectors exists within their ''packaging'' capacity, i.e., they can deliver up to 9 kb of heterologous DNA organized in one or more genes. Furthermore, LVs have a low risk of insertional mutagenesis and oncogenicity as they tend to integrate away from cellular promoters (Cereseto and Giacca, 2004;Ciuffi, 2008) and carry long terminal repeats (LTRs) with low basal and inducible promoter activity. Another benefit of LVs is the absence of pre-existing immunity, which might interfere with the vector's availability. Due to these advantages, LV vectors represent a powerful tool for gene delivery in the context of gene therapy both in vivo and ex vivo.
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However, when used as vectors to deliver genetically encoded tools in the study of neuronal circuits involved in the regulation of energy homeostasis, LVs expose their limitations as viral vectors compared to AAVs, with the disadvantages largely surpassing the advantages. Three studies have highlighted these limitations: Cetin et al. (2006), de Backer et al. (2010a), and Doherty et al. (2011). Generally, when attempting to target a relatively large volume of area such as the hypothalamus or amygdala, these pitfalls can be summarized as follows.
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1. Efficiency: keeping in mind the theoretical and technical complexity of comparing LVs and AAVs, transduction efficiency of LVs is generally low, and only limited areas can be targeted (on this subject, see de Backer et al., 2010b;Rabinowitz et al., 2004;Davis et al., 2002). 2. Vector size: LVs are large viruses. Their viral particles have a diameter of about 100 nm. In the adult rodent brain, the extracellular space is between 40 and 60 nm. Consequently, the diffusion of the LV vector is hindered, thus limiting the spread of virus for efficient transduction. 3. LV vector titers: generally, titers of concentrated LVs are in the range of 5 3 10 7 to 1 3 10 9 transducing units (t.u.)/mL. The injection of a relatively high titer of LV vectors (3.9 3 10 8 t.u./mL) into the lateral hypothalamus (LH) and amygdala (AM) resulted in a very limited amount of transduced cells (de Backer et al., 2010a). On the other hand, the attempt to increase the number of positively transduced neurons by increasing the volume of injected vector dramatically lowered the specificity of the vector itself, resulting in many glial cells being targeted. This is a crucial point to consider as it could profoundly bias the study outcome, especially since glial cells are emerging as important players in the regulation of energy homeostasis.
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Many of these limitations can be overcome using AAV vectors.
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AAVs are small, icosahedral non-enveloped viruses that belong to the family Parvoviridae. To replicate, AAVs require co-infection with a helper virus to complete their replication cycle. Only when a co-infection with either an AdV or a HSV occurs can the AAV complete its replication. The infection of AAVs starts with the interaction between the virion and host cells to mediate internalization of the virus particles. For the AAV serotype 2, this interaction is mainly mediated by the heparan sulfate proteoglycan as a primary receptor (Summerford and Samulski, 1998) and the fibroblast growth factor receptor 1 (FGFR1) (Qing et al., 1999) and the aVb5 integrin (Summerford et al., 1999) functioning as co-receptors. After internalization, the virus is transported into the nucleus, where it releases its genetic material. The AAV genome consists of a single-stranded DNA filament, which is transcriptionally inactive if not converted into a double-stranded DNA molecule. In the host nucleus, the viral DNA can exist in the episomal status or integrated into the host genome. Surprisingly, the AAV shows a preferential site of integration on chromosome 19 (Russell et al., 1994;Walsh et al., 1992). The AAV's genome consists essentially of two genes, each coordinating the genome replication end coding of the structural proteins. Those two genes are eliminated during the generation of the AAV vector and substituted with the genetic material to be transduced (typically a promoter and a gene) (Kotterman and Schaffer, 2014). To date, 12 serotypes of AAV are known (AAV1-AAV12); they present low toxicity and stable long-term transgene expression (McCown, 2005) as well as low immunogenicity (Gonc ¸alves, 2005). AAVs can transduce dividing, as well as quiescent, cells, and different serotypes have been shown to have preferential cell types as hosts. The AAV2 was the first serotype of AAV used for gene delivery in neurons, and it remains one of the most used when targeting the CNS. AAV1, AAV2, and AAV5 have been shown to transduce a high number of neurons within the hypothalamic nuclei, such as the PVN and the SON (Doherty et al., 2011; de Backer et al., 2010a), the LH, and the AM (de Backer et al., 2010b; Blits et al., 2010). Chimeric viruses encoding the capsid of the AAV1 serotype and the genetic material of the serotypes AAV2 and AAV5 have been successfully used to target the arcuate nuclei of the hypothalamus (Sousa -Ferreira et al., 2011;Li et al., 2005). Cell specificity can also be significantly increased by creating recombinant AAV, which packs the genetic material of a specific serotype into the capsid of a different one. Limitations of AAVs include their restricted cloning capacity (less than 5 kb), and as their DNA is single stranded, the necessity to convert their genome into a double-stranded DNA before gene expression can occur.
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When attempting to manipulate a neural circuit by delivering genetically expressed tools, the choice of viral vector should be determined by some key factors, including d the most efficient transduction of the target area, d the highest cell-type specificity of the transduction, d stability of the expression of the inserted modification, and d minimal or no immune response against the viral vector.
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AAVs have been shown to be particularly amenable to the transduction of neurons in the forebrain. Below, we summarize the technical procedures used to successfully target the LH, AM, SON, mediobasal nucleus of the hypothalamus (MDH), PVN, ventromedial hypothalamus (VMH), and nucleus accumbens (NAcc). Bilateral or single injections were performed according to the stereotaxic coordinates listed in Table 1.
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Table 2 shows details of the protocols used to transduce different brain regions directly involved in the regulation of energy homeostasis by using both AAVs and LVs.
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Of note, when using AAVs as the viral vector, the titer of vector can easily be increased to extend the target area without affecting the cell specificity of the vector itself. In de Backer et al. (2010b), the authors directly compared the transduction efficiency of AAV1 and the recombinant LV pseudotyped with the G protein of the vesicular stomatitis virus (VSV-G). A total of 1 3 10 5 t.u. of LVs per site resulted in less than 50 transduced neurons in both the LH and the AM (de Backer et al., 2010b).
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The limited performance of LV as viral vector to target areas in the forebrain was shown in another study comparing five different viral vectors for their efficiency in transducing the suprachiasmatic nucleus of the hypothalamus (Doherty et al., 2011). The authors could detect only a small amount of transduced neurons 4 days after the stereotaxic injection of the LV. The total amount of viral vector used was 5 3 10 5 t.u. in 1 mL. Attempting to increase the number of transduced cells, Doherty et al. (2011) delivered a higher volume (total volume injected was 2.5 mL; total volume was the only parameter that could be adjusted as the concentration of the vector was already the highest possible). Indeed, the number of positively transduced neurons increased but with a concomitant dramatic reduction in the viral vector cell tropism (neurons and glia were equally transduced) (Doherty et al., 2011).
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be rigorously checked and validated. If neurons are the desired target for a certain study, then the chosen viral vector must have a cellular tropism restricted exclusively to neurons. Recent studies have highlighted the emerging role of astrocytes in working together with neurons in orchestrating the fine CNSmediated control of energy metabolism (Buckman and Ellacott, 2014). If this cell population is affected as a consequence of the viral vector, then this loss of specificity would bias the observed phenotype and, ultimately, invalidate the study itself.
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The viral vector should have low or non-immunogenic properties. This characteristic is important for two main reasons: (1) to achieve a successful transduction, the vector should not cause the receiving organism to mount an immunological reaction, which could eliminate the vector itself; and (2) even if there is not a full immunological response against the viral vector, particular attention must be paid to avoid the instauration of a chronic inflammatory state. A tight correlation exists between the onset of obesity and the activation of the hypothalamic inflammatory signal (De Souza et al., 2005;Thaler et al., 2012;Horvath et al., 2010). This could also represent a major confounder in the outcome of the study, especially when long experimental procedures and analyses are involved.
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Acute versus long-term transduction effects should be rigorously evaluated. AAV are single-stranded DNA viruses with the limiting step in the expression of the carried transgene being controlled by the synthesis of the complementary DNA strand. For example, AAV5, which exhibits an extremely high tropism for the neurons of the SON of the hypothalamus, shows long latency times between the injection and the expression of the carried gene. When an acute effect is being studied, this could be a problematic factor.
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It is also important to consider that both LVs and AAVs are able to produce a stable and long-term transduction of the inserted genetic material. Under such conditions, it is possible to speculate that in the presence of other factors (potency of the promoter used, physiology of the host cell, etc.), the expression of the construct being delivered might interfere or compete for the use of reagents and organelles normally dedicated to the phys-iological activity of the host cell. This might represent a serious confounding factor in the interpretation of the experimental data. In the context of using viral vectors to transduce brain regions controlling energy homeostasis, extensive preliminary evaluations should be performed to exclude the effects of the transduction itself on the cell physiology. When hypothalamic neural circuits are transduced, the aspects of two main critical organelles (among others) deserve special attention.
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1. Endoplasmic reticulum (ER) is formed by an interconnected network of flattened, membrane-enclosed sacs called cisternae. The ER serves as one of the main sites for protein synthesis. Within the ER, translation takes place; proteins are folded properly and dispatched to their final destination. Its homeostasis is crucial for cell function, particularly in the hypothalamus, where ER stress plays a causal role in the onset of obesity due to its central function in the development of leptin resistance (Ozcan et al., 2009) and its role in the disruption of the balance of the hypothalamic melanocortin system (the primary hypothalamic anorexigenic circuit) (Schneeberger et al., 2015). When using viral-based systems to transduce neurons controlling energy homeostasis, it would be essential to verify that the expression of the delivered construct does not disrupt ER homeostasis, thus invalidating the entire study. 2. Mitochondria, long considered to be the main production centers of metabolic energy in the form of adenosine trisphosphate (ATP), have recently emerged as a highly dynamic organelles that play a key role in the hypothalamus-mediated regulation of energy homeostasis (Schneeberger et al., 2013). Preliminary experiments and the choice of the most appropriate controls should be carefully planned to verify the neutrality of the transduced construct on mitochondrial function.
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Taken together, the information provided here suggests that when the study of energy homeostasis is one of the end goals and the forebrain regions involved in such regulation are the targets of gene manipulation through viral vectors, AAVs should generally be chosen over LV vector-based strategies because Low transduction efficiency/ specificity when used to target brain nuclei involved in the regulation of energy homeostasis d AAVs have narrow cell tropism: by choosing the adequate AAV serotype, it is possible to target a specific cell population (neurons over glia cells in the aforementioned studies). d compared to LV vectors, AAV have higher transduction efficiency. In different hypothalamic nuclei and AM, injection of a lower number of transducing units of AAV resulted in a higher transduced area when compared to LV vectors. d an increase in the titer of AAV resulted in an increase in the total transduced area without loss of cell tropism. d AAVs are small viruses; their viral particles are between 20 and 30 nm, and this explains the increased transduced area as compared to LV vectors. d AAV size is also the main limiting factor when using them as viral vectors, but their small genome allows the packaging of small-to-medium genetic material. d increasing the titer of AAV preparations is easily achievable, leading to an increase in the transduced area while this is not the case for LVs.
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The advantages and disadvantages of AAVs and LVs as transducing vectors to manipulate the gene expression of brain areas controlling energy metabolism are listed in Tables 3 and 4, respectively.