Surface engineering of lentiviral vectors for gene transfer into gene therapy target cells

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ScienceDirect Surface engineering of lentiviral vectors for gene transfer into gene therapy target cells Camille Le´vy1,2,3,4,5,6, Els Verhoeyen1,2,3,4,5,6,7 and Franc¸ois-Loı¨c Cosset1,2,3,4,5,6 Since they allow gene integration into their host genome, lentiviral vectors (LVs) have strong therapeutic potentials, as emphasized by recent clinical trials. The surface-display of the pantropic vesicular stomatitis virus G glycoprotein (VSV-G) on LVs resulted in powerful tools for fundamental and clinical research. However, improved LVs are required either to genetically modify cell types not permissive to classical VSV-G-LVs or to restrict entry to specific cell types. Incorporation of heterologous viral glycoproteins (gps) on LVs often require modification of their cytoplasmic tails and ligands can be inserted into their ectodomain to target LVs to specific receptors. Recently, measles virus (MV) gps have been identified as strong candidates for LV-retargeting to multiple cell types, with the potential to evolve toward clinical applications. Addresses 1 CIRI, International Center for Infectiology Research, Team EVIR, Universite´ de Lyon, Lyon, France 2 Inserm, U1111, Lyon, France 3 Ecole Normale Supe´rieure de Lyon, Lyon, France 4 Universite´ Claude Bernard Lyon 1, Centre International de Recherche en Infectiologie, Lyon, France 5 CNRS, UMR5308, Lyon, France 6 LabEx Ecofect, Universite´ de Lyon, Lyon, France 7 Inserm, U1065, Centre Me´diterrane´en de Me´decine Mole´culaire (C3M), e´quipe ‘‘controˆle me´tabolique des morts cellulaires’’, Nice 06204, France Corresponding authors: Verhoeyen, Els ([email protected]) and Cosset, Franc¸ois-Loı¨c ([email protected])

Current Opinion in Pharmacology 2015, 24:79–85 This review comes from a themed issue on New technologies Edited by Andrew H Baker and Adrian J Thrasher

http://dx.doi.org/10.1016/j.coph.2015.08.003 1471-4892/# 2015 Elsevier Ltd. All rights reserved.

improved during the last decade. Surface-display of the glycoprotein (gp) G of the vesicular stomatitis virus (VSVG) on LVs [3] (VSV-G-LVs) resulted in a precious tool for fundamental and therapeutic applications because they allow genetic modification (transduction) of a wide range of cell types, which was recently explained by the discovery of the VSV-G receptor, the Low Density Lipoprotein Receptor (LDL-R) [4,5]. Although VSV-G-LVs conferred therapeutic efficacy in different clinical trials [6–8,9] and are now standard tools for gene knockdown or overexpression of genes, improved LV-based tools are needed either to transduce cells not permissive to VSV-G-LVs or to restrict LV entry to a specific cell type (cell tropism), for example, for in vivo applications. Another limitation in the use of VSVG-LVs in vivo is their rapid inactivation by human complement present in the blood [10].

Strategies to ensure efficient recruitment of gp and lentiviral vectors pseudotyping A straightforward strategy consists in substituting the entire wild-type HIV gp by a heterologous gp such as VSV-G. This technology is called pseudotyping and in the case of VSV-G or of the gps from murine leukemia virus (MLV) and of several other enveloped viruses, no structural gp modifications are required for efficient LV surface-display (Table 1). Envelope gps consist of an ectodomain, spiking outside the virion to interact with their cell surface receptor(s) and to fuse with the target cellular membrane, a transmembrane region and a cytoplasmic tail, inside the viral particle, potentially interacting with the LV core. However, co-expression of a given heterologous gp with a lentiviral core will not necessarily result in infectious LVs. The reason for this might be either ‘physical’ for example steric hindrance or misfolding due to lack of adequate viral gp-LV core interaction or ‘functional’, due to a nonoptimal co-localization of the different vector particle components at the assembly site in the producer cells [11,12].

Introduction Lentiviral vectors (LVs) derived from human immunodeficiency virus, have an outstanding therapeutic potential by allowing stable long-term transgene expression through integration properties into the target cell genome [1,2]. The surface design of LVs has been constantly www.sciencedirect.com

Since the motifs implicated in intracellular trafficking are often contained in the gp cytoplasmic tail (CT), their modification proved to be a first choice solution to facilitate incorporation of gps on LVs and to obtain infectious LV particles (Figure 1). For the paramyxoviruses, like Current Opinion in Pharmacology 2015, 24:79–85

80 New technologies

Table 1 Examples of pseudotyped lentiviral vectors with heterologous envelope glycoproteins relying on the natural tropism of these glycoproteins Vector pseudotype Rabies Rabies modified Mokola LCMV

Ross River Ebola Baculovirus GP64 HCV Sendai RD114 modified GALV modified BaEV modified Measles virus

Targeted cells — tissues Neurons Neurons Neurons Glioma and neural stem cells Dendritic cells Glial cells Airway epithelium Porcine airways Hepatocytes Lung Hematopoietic cells Hematopoietic cells Hematopoietic cells Hematopoietic cells

References [44] [17,18,19] [44] [45] [46] [26] [24] [47] [48] [25] [16] [16] [27] [14,28,30,32]

measles virus (MV) [13,14] or Tupaia paramyxovirus (TPMV) [15], shortening the CT of their gps was sufficient to allow LV incorporation. In contrast, for the cat and baboon endogenous retrovirus, RD114 and BaEV, and for the gibbon ape leukemia virus (GALV) glycoproteins, a complete switch of their CT with that of the MLV glycoprotein, which bears intracellular trafficking motifs allowing gp/core co-localization, was required for their incorporation on lentiviral particles [16]. Using similar approaches, the fusion of the VSV-G CT to the Rabies virus glycoprotein (RV-G) transmembrane and ectodomain allowed up to 100-fold gain in RV-G-LV titers [17,18,19]. Importantly, these cytoplasmic tail modifications do not impair the host cells range of these viral gps.

Strategies to retarget gp tropism Targeting of LVs is based on the principle that fusionactivation of a chimeric envelope should be triggered by the interaction of the ligand displayed on the vector surface with its specific receptor on the target cell. Therefore, the addition of ligands or of single chain antibodies (scAb), specific for a cell surface molecule, to the N-terminal domain of retroviral envelope glycoproteins has been investigated in detail. Unfortunately, although specific binding was achieved, the vector-cell membrane fusion was perturbed in most instances. As a consequence, alternative strategies have been designed and based on the separation of the two functions, cell binding and virus-cell fusion, into two separate glycoproteins. Retargeting of the glycoprotein that confers binding to the target cell, leaves in this case the fusion gp ‘untouched’ and fully functional. For instance, Morizono et al. designed a pseudotyped LV able to transduce melanoma tumor cells using this principle [20]. Indeed, they co-pseudotyped LVs with the Sindbis virus gps E1 fusion protein and a mutated E2 protein non-covalently Current Opinion in Pharmacology 2015, 24:79–85

linked to a specific monoclonal antibody directed against melanoma antigen. However, endocytic uptake of the LV particles is required with this system to activate the membrane fusion properties of the E1-E2 proteins through endosomal acidic pH. A similar strategy was applied using the H and F MV envelope proteins, which confer receptor binding (CD46, Nectin-4, SLAM) and membrane-fusion, respectively. These MV-LVs can be retargeted to various cell types using a mutant of the H gp with 4 points mutations that looses binding to its natural receptors (Figure 1). Cell binding is restored by insertion of a given ligand (e.g., epidermal growth factor) or scAb (e.g. anti-CD19) on the H protein, while vector-cell membrane fusion is mediated by the F protein [13]. Finally, an alternative option to structurally modify the envelope is to impact the posttranslational modifications. For example, use of mannosidase inhibitors can alter the tropism of Sindbis virus gp-pseudotyped particles by increasing envelope binding to C-lectins such as DCSIGN [21,22,23].

Pseudotyping with heterologous or engineered viral gps confers novel entry properties to the LVs Selective tropism of vector particles was achieved by taking advantage of the natural tropism of gps from other membrane-enveloped viruses (see Table 1). For instance, the use of surface gps derived from viruses that cause lung infection and infect hosts via the airway epithelia, like Ebola virus or Sendai virus, may prove useful for gene therapy of the human airway [24,25]. Exclusive transduction of retinal pigmented epithelium could be obtained following subretinal inoculations of LV pseudotypes in rat eyes [26]. Importantly, several viral gps could target LVs to the central nervous system (CNS) such as Rabies, Mokola, Lymphocytic choriomeningitis virus (LCMV) or Ross River virus gps that permit transduction of specific cell types in the CNS (Table 1) [26]. Of note, rabies gpsbased LVs mediated axonal uptake and enhanced retrograde transport-mediated gene transfer [17,18,19]. This is an important breakthrough for gene therapy approaches of the multiple diseases affecting the CNS such as lysosomal storage disease, Alzheimer disease, Huntington disease and Parkinson disease [26]. Other viral gps proved to be specifically efficient for LV transduction of hepatocytes or skin (Table 1). Likewise, screening of a large panel of pseudotyped LVs established the superiority of the GALV and RD114 gps for transduction of hematopoietic stem cells (HSCs), progenitor and differentiated hematopoietic cells [16]. Recently, the BaEV glycoprotein exhibited superior transduction of HSCs, even without cytokine stimulation, www.sciencedirect.com

Surface engineering of lentiviral vectors Le´vy, Verhoeyen and Cosset 81

Figure 1

Cytoplasmic tail switch

(a)

Surface unit

Transmembrane unit

MLV-A

Ectodomain

TM

CT

R

RDTR

Ectodomain

TM

CT

R

BaEVTR

Ectodomain

TM

CT

R

Cytoplasmic tail truncation

(b)

Hwt

HΔCT

CT ΔCT

TM

Ectodomain

TM

Ectodomain

Fwt

Ectodomain

TM

FΔCT

Ectodomain

TM ΔCT

(c)

CT

Fusion with a ligand

∗∗∗∗ HΔCT-scAb

ΔCT

TM

scAb/DARPins Current Opinion in Pharmacology

Examples of strategies to engineer envelope glycoproteins. (a) Engineering of the cat endogenous retroviral glycoprotein (RD114) or the baboon endogenous retroviral (BaEV) glycoprotein to allow efficient incorporation on lentiviral vectors (LV). Replacement of cytoplasmic tail of RD114 or BaEV by the cytoplamic tail of MLV-A, resulted in RDTR and BaEVTR chimeras. (b) Schematic presentation of wild-type or mutant F and H gps from the Edmonston measles virus (MV) strain and their cytoplasmic tails truncated forms permitting LV pseudotyping. To ablate the natural receptor recognition, four point-mutation were introduced in the ectodomain, marked by asterisks. (c) Ligand fusion to the ectodomain of MV hemagglutinin to permit cell specific targeting. CT: cytoplasmic tail; TM: transmembrane; R: R-peptide; scAb: single chain monoclonal antibody.

which conserved the ‘stem cell’ character of these cells [27].

specificity since transduction of non-targeted cells is low if not non-existing.

Likewise, the MV-LVs, which mimicked the natural tropism of MV for T, B and dendritic cells allowed efficient transduction of these immune cells [14,28–30] upon cytokine stimulation as well as in their quiescent state (Table 1).

Designed ankyrin repeat proteins (DARPins) were also evaluated as an alternative targeting domain instead of scAb for MV-LVs. DARPins are based on naturally occurring ankyrin repeat proteins (an ubiquitously expressed protein family that mediates specific protein–protein interaction). Combinatorial libraries of DARPins allowed to identify specific high-affinity binders to any kind of target molecule and may be an interesting alternative to some aggregation-prone scFvs [31].

As discussed above, another option is to fuse a ligand or a scFv to the viral envelope gp to restrict the LV-tropism to a specific cell type. MV H gp proved to be easily modified to retarget LVs by introduction of a scFv, as demonstrated by multiple examples (Table 2). This emphasizes the flexibility of this strategy, which is characterized by high www.sciencedirect.com

Both these targeting techniques have been extended to other paramyxovirus envelope gps from Tupaia Current Opinion in Pharmacology 2015, 24:79–85

82 New technologies

Table 2 Examples of ligands fused to measles virus hemagglutinin Ligand scAb CD20 GluR CD105 CD105 CD133 CD133 MHC-II IL13R CD8 CD19 CD30 DARpins Her2neu

Targeted cells — tissues B lymphocytes Neurons Endothelial cells Hematopoietic stem cells Hematopoietic progenitors Glioblastoma stem cells Ag presenting cells Tumor cells T lymphocytes B lymphocytes Tumor cells Tumor cells

Species

References

Human Mouse Human/ mouse Human

[13,35] [49] [40,49]

Human

[41,49]

Human

[51]

Mouse Mouse Human Human Human

[52,53] [54] [42] [35] [55]

Human

[31]

[50]

paramyxovirus (TPMV) and Nipah virus. Because TPMV normally does not infect human cells, ‘detargeting’ from natural receptors was unnecessary. CD20-targeted TPMVLVs were shown to selectively transduce CD20-positive cells, including quiescent human B-cells [15], underlining their value for systemic applications. Very recently, as for MV-gp, Nipah gp was blinded for binding to the natural receptor and retargeted to cell surface receptor of choice (Brendal et al., oral 4 in 18th American Society of Cell and Gene Therapy 2015).

Engineered envelopes can allow specific activation of the target cells Although LVs are able to transduce non-proliferating cells both in vitro and in vivo, some important gene therapy targets are refractory to gene transfer with LVs. This includes, in particular resting HSCs, monocytes and resting T and B lymphocytes [32]. Primary human lymphocytes also normally require stimulation with cytokines to become permissive for transduction with classical VSV-GLVs [32]. To address this issue, there were several attempts to include activating molecules at the LV surface in order to induce signaling and to allow productive LV transduction. In the case of T cells, this strategy was explored by fusing ‘activating’ molecules to the N-terminus of the MLV gp such as anti-CD3, which activates the T cell receptor [33], or IL-7, which allows mild stimulation with conservation of the naı¨ve T-cell phenotype [34]. Coexpression with an ‘escorting’ VSV-G gp was needed to render the LVs fully infectious. Likewise, MV-LVs retargeted to CD20/CD19 surface molecules, achieved through scAb display on MV H, transduced quiescent B-cell. Kneissl et al. demonstrated that the engagement of Current Opinion in Pharmacology 2015, 24:79–85

CD20 or CD19 molecules by such MV-LVs up-regulated activation markers and induced entry into G1b phase of cell cycle [35]. Finally, to overcome the inability of VSVG-LVs to transduce quiescent early hematopoietic progenitor CD34+-cells, LVs were designed to display early acting cytokines on their surface [36]. Co-display of stem cell factor (SCF) with RD114 on the LV surface allowed slight and transient stimulation of HSCs, resulting in efficient HSC gene transfer while preserving their ‘stemness’. Moreover, these vectors (RDTR/SCF-LVs) allowed a selective and long-term transduction of c-kit+ HSCs in vivo in humanized immune system mice [37].

LV optimization for in vivo use A significant concern in the use of VSV-G-LVs for in vivo gene delivery is its inactivation by the human complement cascade. To address this problem, Hwang et al. conducted experimental evolution of VSV-G to increase its resistance to human serum. They identified several surface exposed residues whose mutations increase serum resistance or thermostability. VSV-G variants combining both properties exhibited enhanced gene delivery in the presence of human serum in vivo [38]. The capacity of MV-LVs to transduce quiescent T and B cells highlights the importance of these LV pseudotypes for in vivo gene therapy and immunotherapy [14,30], since the majority of these target cells are quiescent in vivo. However, a major obstacle in the use of H/F-LVs in vivo is that most of the human population is vaccinated against measles. Consequently, MV-LVs are quickly neutralized by antibodies directed against the H gp present in human sera. By mutating major B cell epitopes and modifying the glycosylation pattern in H gp, mutant MV-LVs were engineered and efficiently transduced lymphocytes in the presence of MV antibody-positive human sera [39].

Toward clinical applications of LV pseudotypes Envelope engineering of LVs has already brought several therapeutic applications that were investigated at the preclinical level. Below we present some recent examples. Fanconi anemia (FA) is a bone marrow (BM) failure disorder, which is caused by mutations in FANC genes, involved in DNA repair. Since the HSCs are affected by the disease, their isolation and ex vivo culture induces them into apoptosis; thus, HSCs-targeted vectors might provide a solution. Indeed, low doses of RDTR/SCF-LVs confer efficient targeted FANC gene transfer to CD34+cells in non-fractionated BM from FA patients, allowing to bypass FA CD34+-cell isolation. Therefore, these RDTR/SCF-LVs might completely omit ex vivo handling and simplify gene therapy for many hematopoietic defects by direct in vivo inoculation, which paves the www.sciencedirect.com

Surface engineering of lentiviral vectors Le´vy, Verhoeyen and Cosset 83

way toward in vivo correction of monogenetic diseases such as FA [37]. By introducing a scFv for human endoglin (CD105) into the MV-H protein, a vector targeted to human endothelial cells (aCD105-MV-LV) was generated that specifically transduced human liver sinusoidal or vascular arterial endothelial cells in xenograft mouse models [40]. In addition, aCD105MV-LV allowed delivery of erythropoietin upon systemic injection in a mouse model. Using a similar strategy of scAb surface display, aCD133MV-LVs allowed specific transduction of HSCs. Brendel et al. showed that such vectors could transfer a therapeutic gene into CD34+-cells obtained from patients suffering of X-linked chronic granulomatous disease. aCD133-LVs allow the expression of the missing gp91phox gene into patient CD34+-cells, which restored the defects when engrafted in immunodeficient mice [41]. Transfer of tumor-specific T-cell receptor (TCR) genes into patient T cells is a promising strategy in cancer immunotherapy, which is currently under investigation in clinical trials worldwide. Tumor specificity is provided by an antigen receptor, which can be natural (T-cell receptor; TCR) or engineered (chimeric antigen receptor, CAR). In this context, Zhou et al. generated aCD8-MVLVs that allowed gene delivery using the CD8 surface molecule as receptor for cell entry. Expression of melanoma-reactive TCRs exclusively in CD8+ T cells using this vector significantly enhanced tumor-cell killing compared with conventional gene transfer mediated by a nontargeted LV in a mouse model. This may be due to the activation of the effector functions of CD8+ T cells by the aCD8-MV-LV and to the increase of CD8 surface density, which functions as co-receptor for tumor-cell recognition [42]. Dendritic cells (DCs) are essential antigen-presenting cells for the initiation of cytotoxic T-cell responses and, therefore, are attractive targets for cancer immunotherapy. Odegard et al. developed an integration-deficient LV that was designed to deliver antigen-encoding nucleic acids selectively to hDCs in vivo [43]. This LV utilizes a modified Sindbis virus gp to target hDCs through the Ctype lectin, DC-SIGN [23] and binds the homologue murine receptor SIGNR1. Preclinicals studies in mice showed that this vector exerted antitumor cytotoxic activity in both prophylactic and therapeutic settings. These data support its validity as a therapeutic for cancer in humans as it is investigated in a phase I trial in cancer patients [43]. Concluding, recent progresses in LV surface modification have resulted in tools that are highly specific for target cells in vitro and in vivo and may signify a big step toward clinical applications in vivo. Especially since this avoids www.sciencedirect.com

target cell isolation, ex vivo culture and infection under good manufacturing practice conditions, which is costly and cumbersome.

Conflict of interest statement Nothing declared.

Acknowledgements This work was supported by grants from the ‘Agence Nationale pour la Recherche contre le SIDA et les He´patites Virales’ (ANRS), the ‘Agence Nationale de la Recherche’ (ANR), the European Research Council (ERC2008-AdG-233130 ‘HEPCENT’), the European Community (FP7HEALTH-2007-B/222878 ‘PERSIST’, E-RARE-06-01 ‘GENTHERTHAL’) and the LabEx Ecofect (ANR-11-LABX-0048).

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Current Opinion in Pharmacology 2015, 24:79–85