Review
`Pseudotyping Lentiviral Vectors: When the Clothes
`Make the Virus
`
`Alexis Duvergé 1,2 and Matteo Negroni 1,2,*
`1 Université de Strasbourg, CNRS, Architecture et Réactivité de l’ARN, UPR9002, 67000 Strasbourg, France
`2
`Interdisciplinary Thematic Institute (ITI) InnoVec, Université de Strasbourg, 67000 Strasbourg, France
`* Correspondence: m.negroni@unistra.fr; Tel.: +33-388-417-006
`
`Received: 28 August 2020; Accepted: 11 November 2020; Published: 16 November 2020
`
`Abstract: Delivering transgenes to human cells through transduction with viral vectors constitutes
`one of the most encouraging approaches in gene therapy. Lentivirus-derived vectors are among the
`most promising vectors for these approaches. When the genetic modification of the cell must be
`performed in vivo, efficient specific transduction of the cell targets of the therapy in the absence of
`off-targeting constitutes the Holy Grail of gene therapy. For viral therapy, this is largely determined
`by the characteristics of the surface proteins carried by the vector. In this regard, an important
`property of lentiviral vectors is the possibility of being pseudotyped by envelopes of other viruses,
`widening the panel of proteins with which they can be armed. Here, we discuss how this is achieved
`at the molecular level and what the properties and the potentialities of the different envelope proteins
`that can be used for pseudotyping these vectors are.
`
`Keywords: pseudotyping; lentiviral vectors; envelope proteins; gene therapy
`
`1. Gene Therapy Using Viral Vectors
`
`According to the definition provided by the NIH Genetics Home Reference, gene therapy is an
`experimental technique aimed at treating or preventing a disease by using genes [1]. This can be
`achieved by various means. When the disease is of genetic origin and, particularly, when it is caused
`by a single defective gene, the ultimate goal is replacing the defective gene with a wild-type one.
`This has been possible only recently with the development of powerful genome editing techniques [2–4].
`Although, these are not applicable routinely and alternative approaches are followed, the most common
`of which is the introduction of a gene conferring a dominant wild-type phenotype to the modified
`cell [5]. Whatever the approach followed, gene therapy relies on the use of vectors that allow the
`efficient genetic modification of cells, or tissues, combined with a high specificity for the target cells to
`reduce adverse effects [6]. Introducing exogenous genetic material in cells is efficiently performed by
`cellular “parasites”—phages for bacteria or viruses for eukaryotic cells. In particular, the vast range of
`human viruses provides a large panel of promising tools for vectorization (by transduction) in sight of
`intervention on human cells. How to reprogram human viruses for the purposes mentioned above is a
`major challenge in molecular medicine.
`A main watershed in gene therapy is whether the genetic modification of the cell must be carried
`out ex vivo or in vivo. If the cells’ target for the therapy can be isolated from the patient, modified ex
`vivo, and reinfused in the patient, essentially no specific tropism is required for the vector since the
`cells to modify are the only ones it comes into contact with [7–10]. In this case, the vectors can therefore
`carry pan-tropic envelope proteins such as, for example, the vescicular stomatitis virus (VSV) envelope
`protein G (see below). If, in contrast, the modification of the cells must be carried out in vivo, a high
`specificity for the target cells is required to avoid off-target transduction. The nature of the envelope
`proteins carried by the viral vector is the major determinant for the specificity of transduction.
`
`Viruses 2020, 12, 1311; doi:10.3390/v12111311
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`www.mdpi.com/journal/viruses
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`Most gene therapy clinical trials carried out to date have relied on the use of adeno-associated
`vectors (AAVs) or retroviral vectors, which might be derived from γ-retroviruses or lentiviruses [11].
`Modification of cells in vivo (liver, muscles, central nervous system and retina) has been restricted to
`the use of AAV-derived vectors, while ex vivo approaches (for the genetic modification of T cells and of
`human hematopoietic stem and progenitor cells) have relied on the use of vectors derived from murine
`γ-retroviruses and human lentiviruses. The neat division between clinical trials where AAV vectors
`have been used and those involving retroviral vectors is in part explained by the natural tropism of the
`viruses from which these vectors have been constructed.
`AAV are non-enveloped non-integrative single-stranded DNA viruses of the Parvoviridae family.
`They require coinfection by adenoviruses to replicate and are non-pathogenic for humans. They infect
`replicating as well as quiescent cells and enter into the target cells by interaction with sialic
`acid, heparan sulfate, or galactose present on their surface, and therefore possess a large tropism.
`Differences in the capsid protein of AAV determine cell type-specific preferences and define the
`existence of the eleven serotypes of this virus. For gene therapy, according to the type of target tissue,
`serotypes that naturally target that type of tissue, when such serotypes exist, are the preferred choice
`for building a viral vector. To date, in gene therapy, eight serotypes (1–2 and 4–9) have been used to
`orient viral transduction toward the tissue of interest [12].
`In sharp contrast to AAV, γ-retroviruses and lentiviruses do not present different serotypes
`and no variation in tissue specificity is found for these viruses, which both target blood cells.
`For example, in human immunodeficiency virus (HIV), despite its impressive genetic diversity,
`which is particularly high at the level of its envelope proteins, infection remains essentially restricted
`either to CD4+/CCR5+ or CD4+/CXCR4+ cells. However, an interest of retroviral-derived vectors
`(and therefore of lentiviral-derived vectors as well) comes from the possibility of replacing the original
`envelope proteins with those of other viruses, a process called pseudotyping. In this review article,
`we focus on the perspectives on which pseudotyping lentiviral-derived vectors (LV vectors) open and
`how this is achieved.
`
`2. Lentiviruses and Gene Therapy
`
`Retroviruses are enveloped viruses that integrate in the infected cell. This property has made
`of these viruses the preferred choice for developing vectors when the expression of the transgene
`must be stable or when the transgene must be inherited by the progeny of the transduced cell.
`For these reasons, retroviral vectors have been chosen for the expression of transgenes in hematopoietic
`stem and progenitor cells (HSPCs) and, more recently, they have been used for the transduction of
`peripheral blood cells for the generation of CAR-T cells [13]. Gammaretroviral vectors derived from
`Moloney murine leukemia were used for the earliest gene therapy assays using retroviral vectors.
`They have been successful in the treatment of several primary immunodeficiencies, such as the
`X-linked severe combined immunodeficiency (SCID) or the adenosine deaminase deficiency-induced
`SCID [14–16], and they have been employed in the treatment of the Wiskott–Aldrich syndrome and of
`X-linked chronic granulomatous disease [17–19]. However, γ-retroviral vectors have been progressively
`replaced by the lentiviral vectors (LV vectors), mostly due to the lower levels of induction of the innate
`immune response they trigger [20,21] and, in particular, for biosafety reasons. Indeed, LV vectors
`predominantly integrate in transcription units [22], rather than in regions controlling gene expression
`as promoters and enhancers that are, instead, the preferential sites of integration for gammaretroviral
`vectors [23,24]. This difference has been shown to lead to a lower probability for lentiviruses to cause
`insertional oncogenesis [25,26]. LV vectors have thus been used in most recent trials, always for
`the treatment of blood diseases. Besides treating the same diseases with these new vectors as are
`treated with γ-retroviral vectors mentioned above [27–31], β-thalassemia [32], Fanconi anemia [33],
`metachromatic leukodystrophy [34,35], mucopolysaccharidosis type I [36], adrenoleukodystrophy [37]
`and sickle cell disease [38] have also been made the object of clinical trials using LV vectors.
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`3. Molecular Biology of Lentiviruses
`
`Lentiviruses belong to the subfamily Lentivirinae of retroviruses [39]. They are considered as
`“complex” retroviruses, due to the presence of additional genes, compared to other retroviruses. As all
`retroviruses, they are enveloped integrative viruses. The viral particle is constituted by a spheric matrix
`shell that lies immediately underneath the lipid bilayer, which consists of a patch of the cell membrane
`that is carried over during viral budding from the infected cell [40]. More internally, a fullerene-shaped
`core [41] contains the genomic RNA that is constituted by a single-stranded positive-sense molecule,
`present in two copies in the viral particle, in a dimeric form. Upon infection of the host cell (that occurs
`after recognition of a specific receptor on the surface of the cell) the viral capsid enters the cytoplasm.
`The availability of the nucleotides, to which the capsid is permeable, allows the initiation of reverse
`transcription. This results in the conversion of the genomic RNA into double-stranded DNA which is
`then integrated in the cell genome [42–45].
`Where and when this conversion occurs and is achieved remains a matter of debate. The traditional
`view according to which reverse transcription was completed in the cytoplasm or at the nuclear pore,
`followed by the dismantling of the capsid core and the import of the preintegration nucleoprotein
`complex [46–49], has recently been challenged by the observation of intact or almost-intact cores,
`as well as the detection of ongoing reverse transcription in the nucleus [50]. However, irrespective of
`the form under which the genetic material is imported into the nucleus, the import occurs in an active
`manner, through the interaction of the viral capsid protein p24 with the cellular protein cyclophillin A
`and the cellular splicing factor CSPF6 [51–53]. This interaction leads to the use of the nuclear import
`pathway relying on the pair of nuclear pore proteins Nup153/Nup358 and transportin 3 (TNPO3) [54].
`This complex system allows lentiviruses (in the specific case detailed above, human immunodeficiency
`virus type 1 (HIV-1)) to infect non-replicating cells. This not only allows LV vectors to deliver
`transgenes to cells that naturally do not replicate, but also can be exploited for transducing cells, such as
`HSPCs, that must be kept in a quiescent state to avoid their differentiation and loss of pluripotency.
`Retroviruses as γ-retroviruses are instead unable to enter the nucleus of the infected cell and require
`the disassembly of the nuclear membrane at mitosis for reaching the genome of the infected cell
`for integration.
`Integration is carried out by the viral enzyme integrase with poor sequence specificity for the
`selection of the integration sites, although preferential types of genomic regions (as, for example,
`regions where actively transcribed genes are located, or the proximity with respect to transcription
`start sites) can be defined for the different types of retroviruses [55,56]. The reverse transcription
`product, integrated in the genomic RNA of the infected cell, is called a provirus. The provirus is
`flanked by the terminal repeated regions (LTRs) that contain the viral promoter sequence (see below).
`Transcription from the LTR in 5’ will lead to the synthesis of the new genomic RNA as well as the
`viral proteins required for infection to be continued. At the moment of assembly of the viral particle,
`the dimers of viral genomic RNA will be packaged in the budding particle [57]. The particle will also
`incorporate the envelope proteins at their surface, as detailed below, and be released in the extracellular
`space as an immature particle. Activation of the viral protease in the immature particle, will then lead
`to viral maturation and the production of an infectious virus [40].
`
`4. Molecular Bases for the Making of LV Vectors
`
`4.1. Structure of the Genomic RNA
`
`LV vectors are generally derived from the best characterized lentivirus—human immunodeficiency
`virus type 1 (HIV-1). Lentiviral infection, detailed above, is conceptually composed of two phases.
`Entry and the conversion of the genomic RNA (gRNA) into DNA that will be integrated in the cell’s
`chromosomes are considered as the “early phase” of the infectious cycle. With the exception of entry,
`which depends on the nature of the envelope employed in the viral vector, all the steps of the early
`phase of HIV-1 infection are carried out essentially in the same manner during LV vector-mediated
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`transduction. The late phase, constituted by the production of the gRNA and of the viral proteins,
`is instead absent in the case of LV vector transduction.
`The viral gRNA of HIV-1 is characterized, proceeding from 5’ to 3’, by the terminal repeated
`sequence R, the unique sequence in 5’ U5; then, contiguous to U5, are found the 18 nucleotides
`that constitute the sequence to which the tRNA Lys3 anneals for priming reverse transcription
`(primer binding sequence (PBS)) [58] followed by an untranslated 5’ region that is responsible for the
`dimerization of the gRNA and its packaging in the viral particle [57]. Then, the main three genes
`(gag, pol and env) follow, overlapping the sequences for the auxiliary proteins Vif, Vpr and Vpu, as well
`as the proteins Tat and Rev (Figure 1). Finally, partially overlapping with the 3’ portion of env, the Nef
`coding sequence is found, followed by the unique sequence in 3’ U3, the repeated sequence R and the
`polyA tail [59]. The sequences required for priming the synthesis of the second strand of DNA (3’ and
`central polypurine tracts, -3’ PPT and cPPT, respectively) are located immediately upstream of the
`U3 sequence and in the 3’ end portion of pol, respectively [60,61]. The Rev Responsive Element (RRE)
`sequence that, when bound by the Rev protein allows the export of partially unspliced RNAs from the
`nucleus, is located in the portion of env encoding the gp41 protein [62].
`
`Figure 1. Organization of the human immunodeficiency virus type 1 (HIV-1) genomic RNA. U3,
`unique sequence 3’; R, repeated sequence; U5, unique sequence 5’; Ψ, indicates the packaging and
`dimerization sequences; RRE, Rev responsive element. The PPT sequences as well as the primer
`binding sequence (PBS) region are not shown.
`
`To generate the gRNA of the LV vector, the viral gRNA is modified by removing all the coding
`sequences for the viral proteins and leaving the elements required in cis for genomic RNA packaging,
`reverse transcription and integration. Specifically, the gRNA of the vector must contain: the PBS
`sequence; the 3’ PPT and cPPT sequences; the region (located in the 5’ untranslated portion of the
`genome) responsible for the packaging and dimerization of the genomic RNA; the RRE sequence;
`the repeated terminal sequence R and the sequence U5, which are required for achieving reverse
`transcription and integration [63]. The U3 sequence, instead, is only partially preserved, since a
`large deletion (approximately half of its total length) is made in this sequence [64]. The deletion is
`essential for inactivating, in LV vectors, the promoter activity of U3, generating what are known as
`self-inactivating (SIN) vectors [64]. In natural infections, the U3 sequence is located inside the LTR
`sequences, present at both ends of the proviral DNA (Figure 2A). The U3 sequence located in the 5’ LTR
`contains the promoter that is used to drive the transcription of the genomic RNA. The genomic RNA
`contains only the U3 sequence of the 3’ LTR (Figure 2A). After reverse transcription of this genomic
`RNA, the LTRs are again generated (Figure 2A). In the case of SIN LV vectors, the U3 sequence of the
`3’ LTR carries the deletion (in black in Figure 2B) and it will be this sequence that will be present in
`the genomic RNA. After reverse transcription, this deleted version of U3 will be present in both LTR,
`the 5’ and the 3’ regions (Figure 2B). Transcription is thereby no longer possible from this proviral
`DNA, since the promoter in the U3 sequence in the 5’ LTR is not functional. Taking into account these
`requirements, the gRNA of the LV vector can accommodate up to 8 kb of exogenous sequences.
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`Figure 2. Schematic representation of the structure of the genomic forms of the viral DNA during
`natural infection (panel (A)) or during transfection and transduction by a self-inactivating (SIN)
`lentiviral-derived (LV) vector (panel (B)).
`
`For the generation of the LV vector particle, the plasmid leading to the synthesis of the gRNA
`is cotransfected with transcomplementation plasmids leading to the synthesis of the viral proteins.
`Depending on which generation of LV vectors is considered, the structure of the plasmids varies as
`well as which viral proteins are provided (Figure 3). In this setting, in order to change the tropism of
`the viral vector through pseudotyping, the plasmid encoding the envelope proteins will be chosen to
`carry the desired, non-HIV, envelope protein coding sequences.
`
`Figure 3. The various generations of lentiviral vectors. Top panel. Plasmids employed for constructing first
`generation lentiviral vectors. Three plasmids are employed. (1) A packaging (or transcomplementation)
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`plasmid encodes the Gag, Pol, Vif, Tat, Rev, Nef, Vpr proteins under the control of the Cytomegalovirus
`(CMV) promoter. (2) The envelope protein(s) (the vescicular stomatitis virus (VSV) G protein in the
`example given) is encoded by an expression plasmid under the control of the CMV promoter. (3) Finally,
`the plasmid leading to the synthesis of the genomic RNA (“genomic plasmid”) contains the sequences
`required in cis for the packaging and reverse transcription of the RNA. It also contains the sequence of
`the transgene under the control of an internal promoter (EF1α in the example). The expression of the
`genomic RNA is driven by the 5’ terminal repeated regions (LTR). The sequence U3 in the 3’ LTR is
`partially deleted, inactivating the promoter present in U3 generating self-inactivating (SIN) vectors.
`Middle panel: 2nd generation vectors are built by triple transfection of the producer cells. In this
`generation of LV vectors, the packaging plasmid only encodes Gag, Pol, Tat and Rev, increasing the
`level of biosafety (the auxiliary proteins Vif, Nef and Vpr are absent). Bottom panel, third generation
`of vectors. The packaging plasmid is split in two plasmids, one encoding (under the control of the
`CMV promoter) the Gag and Pol sequences and carrying the Rev responsive element (RRE), the second
`encoding the Rev protein (also under the control of the CMV, in the example given). In the genomic
`plasmid the 5’ LTR sequence is replaced by the sequence of a chimeric LTR where the U3 sequence is
`replaced by that of a heterologous promoter (the Rous Sarcoma Virus-RSV-promoter in the example
`given). Finally, "next generation" vectors have also been elaborated, but since they differ considerably
`from one another, no "synthetic" drawing summarizing them is provided. The main improvement
`consists of the splitting of the coding sequences in a larger number of plasmids, for increasing biosafety.
`
`4.2. Mechanism of Entry in HIV-1
`
`The need for pseudotyping LV vector particles comes, as mentioned above, from the difficulty of
`modifying the mechanism of viral entry of the natural HIV envelope proteins. HIV-1 encodes two
`envelope proteins—gp41 and the gp120. The gp41 is a transmembrane protein that, associating in a
`non-covalent manner to the gp120 (that is located on the external side of the virus), forms an unstable
`heterodimer [65]. Three of these heterodimers associate to form a trimer of dimers that constitute
`the viral spike [65]. Viral entry occurs by fusion of the cell and viral membranes, carried out by the
`viral envelope proteins. For this, the spike interacts, through the gp120 component, with the natural
`HIV-1 receptor, the CD4 molecule [66,67]. This triggers a conformational change that leads to the
`generation in the gp120 of a binding site for the HIV coreceptor, generally the transmembrane protein
`CCR5, or CXCR4 [68–72]. This second interaction is responsible for another structural rearrangement
`of the gp120/gp41 dimer that releases the gp41 from the interaction with the gp120 [73]. The gp41 that
`was maintained in a metastable state by the interaction with the gp120 inserts its highly hydrophobic
`N-terminal portion, called “fusion peptide”, in the internal portion of the spike, in the membrane
`of the target cell [74]. Once this has occurred, the gp41 folds back on itself to reach the most stable
`conformation possible. This brings the fusion peptide (still inserted in the cell membrane) in proximity
`of the viral membrane leading to the fusion of the membranes [75–78] and to the creation of a pore
`that, once enlarged, allows the entry of the viral core into the cytoplasm. Because of the complex
`series of conformational transitions required for the functionality of the envelope, the interactions
`between the two Env proteins must be based on highly unstable equilibria that are extremely difficult
`to retain if one wishes to modify this system, in order to redesign the tropism of the virus. Therefore,
`changing the tropism of a HIV-derived LV vectors is a fairly difficult goal to achieve through the
`modification of the natural HIV envelope proteins. However, the relative ease with which a LV vector
`can be efficiently pseudotyped by exogenous viral envelope proteins provides alternative solutions to
`bypass these difficulties.
`
`4.3. Mechanism of Recruitment of HIV-1 Envelope Proteins on the Surface of the Virus: Bases for Pseudotyping
`
`The HIV-1 particle is enveloped by the plasma membrane of the cell from which the virus has
`budded. Consequently, the lipid and protein compositions of the viral membrane reflect that of the
`infected cell at the site of budding. The peculiar composition of lipids and proteins of the viral particle
`with respect to that of the cell, suggests that viral budding occurs in specific regions of the membrane
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`with a particular lipid composition. To be incorporated in the budding viral particle a protein must be
`addressed to the cell compartment where viral budding occurs [79]. These observations set the bases
`to conceive the possibility of pseudotyping lentiviral particles.
`HIV-1 assembles at lipid rafts, areas of the membrane enriched in cholesterol and sphingolipids.
`This lipid composition tends to be enriched in glycosylphosphatidylinositol-anchored proteins at
`the site of budding [80]. Potentially, any protein with a high “affinity” for lipid rafts has a higher
`probability than the average protein to be found on the viral particle. This strategy allows the virus
`to reduce immunogenicity during natural infections and, consequently, also leads to the generation
`of poorly immunogenic HIV-derived LV vector particles. Assembling at lipid rafts indeed provides
`a lipid membrane of the viral particle enriched in proteins such as CD46, CD55 and CD59 [81–84]
`which are known to inhibit complement activation [85–87]. Accordingly, when the virus is produced
`in glycosylphosphatidylinositol-anchors deficient cells, it becomes sensitive to degradation by the
`immune system [88].
`In HIV-1, the envelope proteins are recruited at lipid rafts through the interaction between the
`cytoplasmic tail of the gp41 and the precursor polyprotein Pr55 Gag, which is localized at the lipid
`rafts thanks to the myristoyl group that is present at its N-terminus [89]. It also has been shown that
`the acylation of the transmembrane domains of proteins was sufficient to address these proteins to
`the lipids raft [90,91]. Acylated proteins potentially prevail in viral particles that bud from lipid raft
`rich areas. This characteristic of acylated proteins provides a “tool” to induce pseudotyping in LV
`vectors. Accordingly, VSV glycoproteins are acylated [92], as well as the E2 envelope glycoprotein
`of alphaviruses such as Semliki forest virus and Sindbis Virus [93,94]. All these envelope proteins
`efficiently pseudotype LV vectors. For other viral envelope proteins, such as the rabies ones for instance,
`the molecular mechanism leading to pseudotyping LV vectors is known in much less detail, but it
`is logical to expect that, also in this case, pseudotyping occurs through addressing these proteins to
`lipid rafts.
`
`5. Dressing LV Vectors (Pseudotyping)
`
`Pseudotyping LV vectors with envelope proteins of different viruses allows combining the
`properties of lentiviruses with those of viral entry of other viruses. The envelope proteins of several
`types of viruses have been shown to be able to pseudotype LV vectors. Among these viruses,
`some possess a large tropism and therefore the use of their proteins for treatments in vivo cannot
`be envisaged. However, some of these proteins can be used as starting platforms for engineering
`variants that specifically target a desired cell population. Envelope proteins from other viruses,
`instead, present a tropism restricted to certain types of cells (neurons, for example), and can be
`employed “opportunistically” when these cell types constitute the target of the intervention strategy.
`Envelope proteins from several viruses have been described to successfully pseudotype LV vectors.
`Those for which the molecular mechanism has been elucidated in more detail fall into three viral
`families and are presented here (Figure 4 and Table 1).
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`Figure 4. Outline of the mechanisms of viral entry by the main envelope proteins that can be used to
`pseudotype LV vectors. ECM: Extracellular Medium; MVB: Multivesicular Body.
`
`Table 1. Overview table summarizing the main characteristics of the pseudotypes discussed in
`this review.
`
`Original Virus
`
`Pseudotype
`
`Main Characteristics of
`the Pseudotyped LV
`
`Natural Cell
`Tropism
`
`Vesicular
`Stomatitis virus
`
`VSV-G
`
`Quasi-universal tropism,
`high efficiency
`
`Rabies virus
`
`RabV-G
`
`Natural ability to efficiently
`targets neurons
`
`Broad
`
`Neurons
`
`Measle virus
`
`Nipah virus
`
`H/F
`
`G/F
`
`High efficiency, tolerant to
`peptide insertion, can be
`neutralized by vaccines
`
`Low prevalence: low
`neutralization hazard
`
`B cells, T cells,
`Epithelial cells,
`Dendritic cells,
`HSPC
`Pericytes, tumor
`endothelium
`
`Chickungunya
`virus
`
`E1/E2
`
`Versatile basis for
`engineering/reprograming
`
`Sindbis virus
`
`E1/E2
`
`Versatile basis for
`engineering/reprograming,
`Low immunogenicity
`
`Broad
`
`Broad
`
`Receptor
`
`LDL-R
`
`nAChR,
`CD56,
`p75NTR,
`mGluR2
`
`CD46,
`SLAM,
`nectin-4
`
`EphrinB2,
`EphrinB4
`
`PHB1,
`Mxra8,
`integrins,
`Heparan
`sulfates
`
`67LR,
`NRAMP2
`
`Transduction
`Efficiency
`
`References
`
`High
`
`[95,96]
`
`Up to 50%
`
`[97–100]
`
`Up to 50–70%
`
`[101–108]
`
`20–40%
`
`[109,110]
`
`Low on
`non-adherent
`cells, high on
`adherent cells
`(related to
`VSV-G)
`
`[111–114]
`
`Variable
`
`[115,116]
`
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`5.1. Rhabdoviruses: Clathrin-Dependent Endocytosis
`
`5.1.1. Vescicular Stomatitis Virus
`
`Pseudotyping LV vectors by the vescicular stomatitis virus envelope glycoprotein G is the most
`common approach for creating cell lines [117]. VSV is an enveloped virus from the Rhabdoviridae family.
`It expresses the G glycoprotein on the envelope surface. The first hypothesis to explain the large tropism
`of the virus suggests that it might use not only a specific, widespread, receptor but that it possibly
`also uses alternative receptors. The first receptor described for VSV-G were the phosphatidylserines,
`phospholipids that are a main components of plasma membranes [118,119] present at the surface
`of almost all cell types [119]. However, more recent work showed that treatment with annexin V,
`a specific ligand for phosphatidylserines, did not inhibit infection by VSV [120]. In addition, the same
`work showed an absence of correlation between the content of phosphatidylserines in the plasma
`membrane and the efficacy of infection by VSV [120]. In 2013 it was demonstrated that the main
`receptor promoting VSV entry are members of the LDL-Receptor (LDL-R) family [95]. These receptors
`are involved in the regulation of the homeostasis of cholesterol in mammalian cells and are ubiquitously
`expressed [121,122]. Once bound to the cell membrane, the VSV envelope protein VSV-G triggers
`clathrin-dependent endocytosis [123], typical of the Rhabdoviridae family, followed by pH-dependent
`fusion of endosomal and viral membranes [124,125], leading to the release of the capsid in the cell,
`although it is still debated whether membrane fusion occurs in the early endosome or in the late
`endosomes/multivesicular bodies [126,127]. To begin fusion, a conformational change of the G protein
`is required first to anchor the virus into the cell membrane and then to operate a physical connection
`between the two lipid bilayers that ultimately allows membrane fusion [128].
`The quasi-universal tropism provided by the glycoproteins G of VSV makes it difficult to conceive
`a safe manner for the systemic inoculation of these vectors in patients, because of obvious problems
`related to off-target delivery. When the natural biodistribution that follows the systemic administration
`of vectors is favorable, as for example when the liver is the target of the therapy, LV vectors pseudotyped
`by the VSV envelope protein G have proved to be successful for in vivo treatment, as for the case
`of the induction of the expression of the coagulation factor IX (FIX) in mice and hemophilic dog
`models [129,130]. Alternatively, their local administration can be considered in vivo, as it has been
`shown for colorectal administration in mouse models [131]. However, to date, pseudotyping LV
`vectors by the VSV envelope protein G for gene therapy is employed, in the majority of the cases,
`for transduction ex vivo [132,133].
`
`5.1.2. Rabies Virus
`
`Rabies viruses (RVs) are negatively stranded RNA Rhabdoviruses, with a natural tropism for
`neurons. Accordingly, the use of their envelope proteins for pseudotyping LV vectors is strictly
`related to intervention on these cells. RVs infect neurons through their terminal axons and spread
`through the synapses in a retrograde direction, a feature that is maintained when RV-pseudotyped
`LV vectors are used [134]. Recognition of the receptor is ensured by the G protein, which interacts
`with a panel of different receptors, all expressed on neurons. After receptor recognition, RV particles
`are endocytosed following a clathrin-dependent uptake [135]. The internalized vesicles then fuse
`with the early endosomes, as a consequence of the acidification of the endosome, with a VSV-like
`mechanism [123] (Figure 3).
`The first receptor described for RV was the nicotinic acetylcholine receptor (nAChR) [97].
`This receptor is present at a high density in neuromuscular junctions [136]. Another receptor
`used by RV is CD56 (or NCAM) [98], involved in the adhesion of neural cells, the development
`of neurites and the synapses’ plasticity. However, CD56 is also abundant on natural killer cells,
`raising a concern about the specificity of its use. RVs have also been shown to interact with the
`nerve growth factor receptor (NGFR) superfamily, the p75NTR (Low-affinity Nerve Growth Factor
`Receptor: LNGFR) [99]. However, despite this interaction, infection is limited to around 20% of
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`neurons, while more than 80% are p75NTR positive [137]. These results tend to show that the LNGFR
`is not the most important receptor for RV uptake. Finally, mGluR2, abundant in the central nervous
`system [100], has also been described as a receptor for RV. Indeed, it has been observed that RV and
`mGluR2 are internalized into cells and transported to early and late endosomes in close association,
`suggesting their functional interaction.
`In conclusion, the efficient transduction by the RV G glycoprotein involves a wide panel of
`receptors, but is strictly limited to neurons. This characteristic can be exploited to transduce specifically
`neurons. Indeed, it has been shown that an injection of RV-pseudotyped LV vectors directly in the
`muscle can lead to gene transfer in the spinal cord motoneurons while, under the same conditions,
`a vector pseudotyped wit