Variable correction of Artemis deficiency by I-Sce1-meganuclease-assisted homologous recombination in murine hematopoietic stem cells.

The correction of genetic mutations by homologous recombination is an attractive approach to gene therapy. We used the DNA double-strand breaks introduced by the site-specific endonuclease I-Sce1 as a means of increasing homologous recombination of an exogenous DNA template in murine hematopoietic stem cells (mHSCs). To develop this approach, we chose an Artemis knockout (Art(-/-)) mouse in which exon 12 of the Artemis gene had been replaced by an I-Sce1 recognition site. The I-Sce1 enzyme and the Artemis correction template were each delivered by a self-inactivating (SIN)-integrase-defective lentiviral vector (SIN-IDLV-CMV-ISce1 and SIN-IDLV-Art, respectively). Transduction of Art(-/-) mHSCs with the two vectors successfully reverted the Art(-/-) phenotype in 2 of our 10 experiments. Even though the potential for genotoxicity has yet to be evaluated, this new approach to gene editing appears to be promising. Improving the efficacy of this approach will require further technical work.

HIV-derived vectors for therapy and vaccination against HIV.

Despite being at the origin of one of the world's most devastating public health concerns, the Human Immunodeficiency Virus (HIV) has properties that can be harnessed for therapeutic purposes. Indeed, the ability of HIV to efficiently deliver its genome into the nuclear compartment makes it an ideal vector for gene delivery into target cells. The design of so-called HIV-derived vectors, or more generally lentiviral vectors (LVs), consists in keeping only the parts of the virus that ensure efficient nuclear delivery while entirely removing all coding sequences that contribute towards the replication and pathogenesis of the virus: as a result, the vector genome is composed of less than 10% of the original virus genome and exclusively cis-active sequences. Proteins required for the formation of the lentivector particles and for the early steps of viral replication (including Gag- and Pol-derived proteins) are provided in trans. HIV-derived vectors are thus non-replicative virus shells that deliver genes of interest into target cells with high efficiency. Undoubtedly, there is a hopeful twist of fate in our fight against AIDS, which consists in using these vectors to achieve gene therapy and vaccination against HIV itself. This review summarises the current generation of LVs with a special focus on vaccine applications against AIDS. Preclinical data are very encouraging and efforts are ongoing to optimise these vectors, to increase their safety and improve their immunogenicity.

A haplotype of the human CXCR1 gene protective against rapid disease progression in HIV-1+ patients.

Chemokines and their receptors are key factors in the onset and progression of AIDS. Among them, accumulating evidence strongly indicates the involvement of IL-8 and its receptors, CXCR1 and CXCR2, in AIDS-related conditions. Through extensive investigation of genetic variations of the human CXCR1-CXCR2 locus, we identified a haplotype of the CXCR1 gene (CXCR1-Ha) carrying two nonsynonymous single nucleotide polymorphisms, CXCR1_300 (Met to Arg) in the N terminus extracellular domain and CXCR1_142 (Arg to Cys) in the C terminus intracellular domain. Transfection experiments with CXCR1 cDNAs corresponding to the CXCR1-Ha and the alternative CXCR1-HA haplotype showed reduced expression of CD4 and CXCR4 in CXCR1-Ha cells in human osteosarcoma cells as well as in Jurkat and CEM human T lymphocytes. Furthermore, the efficiency of X4-tropic HIV-1(NL4-3) infection was significantly lower in CXCR1-Ha cells than in CXCR1-HA cells. The results were further confirmed by a series of experiments using six HIV-1 clinical isolates from AIDS patients. A genetic association study was performed by using an HIV-1(+) patient cohort consisting of two subpopulations of AIDS with extreme phenotypes of rapid and slow progression of the disease. The frequency of the CXCR1-Ha allele is markedly less frequent in patients with rapid disease onset than those with slow progression (P = 0.0003). These results provide strong evidence of a protective role of the CXCR1-Ha allele on disease progression in AIDS, probably acting through modulation of CD4 and CXCR4 expression.

Blood-brain barrier-specific properties of a human adult brain endothelial cell line.

Establishment of a human model of the blood-brain barrier has proven to be a difficult goal. To accomplish this, normal human brain endothelial cells were transduced by lentiviral vectors incorporating human telomerase or SV40 T antigen. Among the many stable immortalized clones obtained by sequential limiting dilution cloning of the transduced cells, one was selected for expression of normal endothelial markers, including CD31, VE cadherin, and von Willebrand factor. This cell line, termed hCMEC/D3, showed a stable normal karyotype, maintained contact-inhibited monolayers in tissue culture, exhibited robust proliferation in response to endothelial growth factors, and formed capillary tubes in matrix but no colonies in soft agar. hCMEC/D3 cells expressed telomerase and grew indefinitely without phenotypic dedifferentiation. These cells expressed chemokine receptors, up-regulated adhesion molecules in response to inflammatory cytokines, and demonstrated blood-brain barrier characteristics, including tight junctional proteins and the capacity to actively exclude drugs. hCMEC/D3 are excellent candidates for studies of blood-brain barrier function, the responses of brain endothelium to inflammatory and infectious stimuli, and the interaction of brain endothelium with lymphocytes or tumor cells. Thus, hCMEC/D3 represents the first stable, fully characterized, well-differentiated human brain endothelial cell line and should serve as a widely usable research tool.

Brain engraftment of autologous macrophages transduced with a lentiviral flap vector: an approach to complement brain dysfunctions.

Transplantation of ex vivo gene-corrected autologous cells represents an attractive therapeutic approach for brain diseases. Among the cells of the central nervous system, brain macrophages are promising candidates due to their role in tissue homeostasis and their implication in several neurological diseases. Up to now, gene transfer into macrophages has proven difficult by most currently available gene delivery methods. We describe herein, an efficient transduction of rat bone marrow-derived and brain macrophages with an HIV-1-derived vector containing a central DNA flap and encoding the GFP reporter gene (TRIP-DeltaU3-GFP). In primary cultures of macrophages our results show that more than 90% of the cells were transduced by the TRIP vector and that GFP expression remained stable for 1 month without cytopathic effect. In vivo, transplants of transduced macrophages into the striatum of adult rats exhibited long-term expression of GFP up to 3 months. Transduced macrophages were observed around the brain injection site and exhibited the brain macrophage/microglia phenotype. There was no significant sign of astrogliosis around the graft. These results confirm the potential of lentiviral vectors for efficient and stable ex vivo transduction of macrophages. Moreover, transduced autologous macrophages appear as a valuable vehicle for long-term and localized gene expression into the brain.

IL-7 differentially regulates cell cycle progression and HIV-1-based vector infection in neonatal and adult CD4+ T cells.

Differences in the immunological reactivity of umbilical cord (UC) and adult peripheral blood (APB) T cells are poorly understood. Here, we show that IL-7, a cytokine involved in lymphoid homeostasis, has distinct regulatory effects on APB and UC lymphocytes. Neither naive nor memory APB CD4(+) cells proliferated in response to IL-7, whereas naive UC CD4(+) lymphocytes underwent multiple divisions. Nevertheless, both naive and memory IL-7-treated APB T cells progressed into the G(1b) phase of the cell cycle, albeit at higher levels in the latter subset. The IL-7-treated memory CD4(+) lymphocyte population was significantly more susceptible to infection with an HIV-1-derived vector than dividing CD4(+) UC lymphocytes. However, activation through the T cell receptor rendered UC lymphocytes fully susceptible to HIV-1-based vector infection. These data unveil differences between UC and APB CD4(+) T cells with regard to IL-7-mediated cell cycle progression and HIV-1-based vector infectivity. This evidence indicates that IL-7 differentially regulates lymphoid homeostasis in adults and neonates.

The HIV-1 DNA flap stimulates HIV vector-mediated cell transduction in the brain.

During HIV-1 reverse transcription, central initiation of the plus-strand DNA at the central polypurine tract (cPPT) and central termination at the central termination sequence (CTS) lead to the formation of a three-stranded DNA structure: the HIV-1 central DNA flap. We recently reported that the DNA flap acts as a cis-active determinant of HIV-1 genome nuclear import. Commonly employed HIV-1-derived vectors (HR vectors) lack the central DNA flap. Here we report that the insertion of this DNA flap sequence into HR vectors (TRIP vectors) improves gene transduction in neural cells, ex vivo and in vivo, in rat brain. When neural cells are exposed to increasing concentrations of TRIP vector particles, transgene expression correlates with the dose of vector. This effect contrasts with the plateau observed when using an HR vector. We further demonstrate that the increase of in vivo transduction efficiency obtained with TRIP vectors is due to the stimulation of their genome nuclear import.

Enhanced transgene expression in cord blood CD34(+)-derived hematopoietic cells, including developing T cells and NOD/SCID mouse repopulating cells, following transduction with modified trip lentiviral vectors.

The recent development of lentivirus-derived vectors is an important breakthrough in gene transfer technology because these vectors allow transduction of nondividing cells such as hematopoietic stem cells (HSC), due to an active nuclear import of reverse-transcribed vector DNA. We recently demonstrated that addition of the central DNA flap of HIV-1 to an HIV-derived lentiviral vector strikingly increases transduction of CD34(+) cells. We now describe improvements of the transduction protocol designed to preserve HSC properties and two modifications of the previously described TRIP-CMV vector. First, deletion of the enhancer/promoter of the 3' LTR in the TRIP-CMV vector resulted in a safer vector (TRIPDeltaU3-CMV) with conserved transduction efficiency and increased EGFP transgene expression. Second, the original internal CMV promoter was replaced with the promoter for the ubiquitously expressed elongation factor 1alpha (EF1alpha). This promoter substitution resulted in a significantly more homogeneous expression of the EGFP transgene in all hematopoietic cell types, including CD34(+)-derived T lymphocytes, in which the CMV promoter was inactive, and NOD/SCID mouse repopulating cells. We thus present here an HIV-derived lentiviral vector, TRIPDeltaU3-EF1alpha, which can very efficiently transduce human cord blood HSC and results in high long-term transgene expression in CD34(+)-derived T, B, NK, and myeloid hematopoietic cells.

Lentivirus-mediated gene transfer in primary T cells is enhanced by a central DNA flap.

Retroviral vectors have become the primary tool for gene delivery into hematopoietic cells, including T lymphocytes. Lentiviral vectors offer an advantage over Moloney murine leukemia virus (MuLV) vectors because of their ability to translocate across an intact nuclear membrane and integrate into the genome of nonproliferating cells. We have recently demonstrated that a central strand displacement event, controlled by the central polypurine tract (cPPT) and the central termination sequence (CTS), results in the formation of a central DNA flap which acts as a cis-determinant of HIV-1 genome nuclear import. Here, we show that insertion of this DNA determinant in a classical lentiviral vector resulted in a significantly higher level of transduction in activated T cells (51 +/- 12.7% versus 15 +/- 1.4%). CD4(+) and CD8(+) T cells were transduced at equivalent levels. Importantly, freshly isolated T cells stimulated only during the 12-h transduction period could be efficiently transduced with this new flap-containing lentiviral vector, but not with the parental lentiviral vector nor an MuLV vector. Transgene expression in the flap-containing lentiviral vector, under the control of either an internal cytomegalovirus or the elongation factor-1 alpha (EF1 alpha) promoter, was significant and expression remained elevated in resting T cells. Thus, this system allows stable expression of transgenes in T lymphocytes following a short ex vivo transduction protocol.

The maturation of dendritic cells results in postintegration inhibition of HIV-1 replication.

Maturation of dendritic cells (DC) is known to result in decreased capacity to produce HIV due to postentry block of its replicative cycle. In this study, we compared the early phases of this cycle in immature DC (iDC) and mature DC (mDC) generated from monocytes cultured with GM-CSF and IL-4, trimeric CD40 ligand (DC(CD40LT)), or monocyte-conditioned medium (DC(MCM)) being added or not from day 5. Culture day 8 cells exposed to X4 HIV-1(LAI) or R5 HIV-1(Ba-L) were analyzed by semiquantitative R-U5 PCR, which detects total HIV DNA. CXC chemokine receptor 4(low) (CXCR4(low)) CCR5(+) iDC harbored similar viral DNA amounts when exposed to either strain. HIV-1(LAI) entered more efficiently into DC(CD40LT) or DC(MCM) with up-regulated CXCR4. CCR5(low) DC(CD40LT) still allowed entry of HIV-1(Ba-L), whereas CCR5(-) DC(MCM) displayed reduced permissivity to this virus. Comparing amounts of late (long terminal repeat (LTR)-gag PCR) and total (R-U5 PCR) viral DNA products showed that HIV-1(Ba-L) reverse transcription was more efficient than that of HIV-1(LAI), but was not affected by DC maturation. Southern blot detection of linear, circular, and integrated HIV DNA showed that maturation affected neither HIV-1 nuclear import nor integration. When assessing virus transcription by exposing iDC to pNL4-3.GFP or pNL4-3.Luc viruses pseudotyped with the G protein of vesicular stomatitis virus (VSV-G), followed by culture with or without CD40LT or MCM, GFP and luciferase activities decreased by 60-75% in mDC vs iDC. Thus, reduced HIV replication in mDC is primarily due to a postintegration block occurring mainly at the transcriptional level. We could not relate this block to altered expression and nuclear localization of NF-kappa B proteins and SP1 and SP3 transcription factors.

The human immunodeficiency virus type-1 central DNA flap is a crucial determinant for lentiviral vector nuclear import and gene transduction of human hematopoietic stem cells.

Gene transfer in human hematopoietic stem cells (HSCs) has great potential for both gene therapy and the understanding of hematopoiesis. As HSCs have extensive proliferative capacities, stable gene transfer should include genomic integration of the transgene. Lentiviral vectors are now preferred to oncoretroviral vectors especially because they integrate in nondividing cells such as HSCs, thereby avoiding the use of prolonged cytokine stimulation. Human immunodeficiency virus type-1 (HIV-1) has evolved a complex reverse transcription strategy including a central strand displacement event controlled in cis by the central polypurine tract (cPPT) and the central termination sequence (CTS). This creates, at the center of HIV-1 linear DNA molecules, a 99-nucleotide-long plus-strand overlap, the DNA flap, which acts as a cis-determinant of HIV-1 genome nuclear import. The reinsertion of the DNA flap sequence in an HIV-derived lentiviral vector promotes a striking increase of gene transduction efficiency in human CD34(+) hematopoietic cells, and the complementation of the nuclear import defect present in the parental vector accounts for this result. In a short ex vivo protocol, the flap-containing vector allows efficient transduction of the whole hierarchy of human HSCs including both slow-dividing or nondividing HSCs that have multiple lymphoid and myeloid potentials and primitive cells with long-term engraftment ability in nonobese diabetic/severe combined immunodeficiency mice (NOD/SCID).

HIV-1 genome nuclear import is mediated by a central DNA flap.

HIV-1 and other lentiviruses have the unique property among retroviruses to replicate in nondividing cells. This property relies on the use of a nuclear import pathway enabling the viral DNA to cross the nuclear membrane of the host cell. In HIV-1 reverse transcription, a central strand displacement event consecutive to central initiation and termination of plus strand synthesis creates a plus strand overlap: the central DNA flap. We show here that the central DNA flap acts as a cis-determinant of HIV-1 DNA nuclear import. Wild-type viral linear DNA is almost entirely imported into the nucleus where it integrates or circularizes. In contrast, mutant viral DNA, which lacks the DNA flap, accumulates in infected cells as unintegrated linear DNA, at the vicinity of the nuclear membrane. Consistently, HIV-1 vectors devoid of DNA flap exhibit a strong defect of nuclear import, which can be corrected to wild-type levels by reinsertion of the DNA flap sequence.

Predominance of CCR5-dependent HIV-1 subtype E isolates in Cambodia.

To investigate the genetic and biologic features of HIV-1 strains circulating in Cambodia, viruses from 95 HIV-1-seropositive individuals were subtyped by heteroduplex mobility assay (HMA) and 23 were further analyzed for their biologic characteristics. Eighty-nine individuals were clearly infected by HIV-1 subtype E. The other six samples were sequenced, together with 17 HMA subtype E samples. All but one of the 23 Cambodian env sequences clustered with previously described Thai and Vietnamese subtype E sequences, bearing a GPGQ motif at the tip of the V3 loop; the last had a GPGR motif and was phylogenetically equidistant from Asian and African subtype E viruses. Nonsyncytium-inducing, CCR5-dependent viruses predominated in patients of clinical stage B even in some with a high viral load and were detected in about 50% of the patients of stage C. All syncytium-inducing strains, mostly from AIDS patients, used both CCR5 and CXCR4. The presence of syncytium-inducing viruses did not correlate with the plasma viral load. These data show that CCR5-dependent HIV-1 subtype E is currently predominant in Cambodia. The analysis of clinical and virologic markers strongly supports the idea that dynamics of the viral population during subtype E infection in Southeast Asia is similar to that of subtype B infection in Europe and the United States.

PIP: The National AIDS Control and Prevention Program of the Cambodian ministry of health has reported that the prevalence of HIV-1 infection among blood donors in Cambodia increased from less than 1% in 1991 to 4% in 1996, and that 39.3% of prostitutes, 7.1% of military personnel, 3.2% of pregnant women, and 5.2% of tuberculosis patients were infected in 1997. Findings are presented from an investigation of the genetic and biological features of HIV-1 strains in Cambodia. Viruses from 95 HIV-1-seropositive individuals were subtyped by heteroduplex mobility assay (HMA) and 23 were further analyzed for their biologic characteristics. 89 people were clearly infected with HIV-1 subtype E. The other 6 samples, however, were sequenced together with 17 HMA subtype E samples. All but 1 of these latter 23 Cambodian env sequences clustered with previously described Thai and Vietnamese subtype E sequences bearing a GPGQ motif at the tip of the V3 loop, with the last having a GPGR motif and being phylogenetically equidistant from Asian and African subtype E viruses. Nonsyncytium-inducing, CCR5-dependent viruses predominated in patients of clinical stage B, and were detected in about half of the stage C patients. All syncytium-inducing strains, mostly from AIDS patients, used both CCR5 and CXCR4. The presence of syncytium-inducing viruses did not correlate with the plasma viral load. These data show that CCR5-dependent HIV-1 subtype E currently predominates in Cambodia. The dynamics of the viral population during subtype E infection in Southeast Asia appear to be similar to that of subtype B infection in Europe and the US.

Antibodies to several conformation-dependent epitopes of gp120/gp41 inhibit CCR-5-dependent cell-to-cell fusion mediated by the native envelope glycoprotein of a primary macrophage-tropic HIV-1 isolate.

The beta-chemokine receptor CCR-5 is essential for the efficient entry of primary macrophage-tropic HIV-1 isolates into CD4(+) target cells. To study CCR-5-dependent cell-to-cell fusion, we have developed an assay system based on the infection of CD4(+) CCR-5(+) HeLa cells with a Semliki Forest virus recombinant expressing the gp120/gp41 envelope (Env) from a primary clade B HIV-1 isolate (BX08), or from a laboratory T cell line-adapted strain (LAI). In this system, gp120/gp41 of the "nonsyncytium-inducing," primary, macrophage-tropic HIV-1BX08 isolate, was at least as fusogenic as that of the "syncytium-inducing" HIV-1LAI strain. BX08 Env-mediated fusion was inhibited by the beta-chemokines RANTES (regulated upon activation, normal T cell expressed and secreted) and macrophage inflammatory proteins 1beta (MIP-1beta) and by antibodies to CD4, whereas LAI Env-mediated fusion was insensitive to these beta-chemokines. In contrast soluble CD4 significantly reduced LAI, but not BX08 Env-mediated fusion, suggesting that the primary isolate Env glycoprotein has a reduced affinity for CD4. The domains in gp120/gp41 involved in the interaction with the CD4 and CCR-5 molecules were probed using monoclonal antibodies. For the antibodies tested here, the greatest inhibition of fusion was observed with those directed to conformation-dependent, rather than linear epitopes. Efficient inhibition of fusion was not restricted to epitopes in any one domain of gp120/gp41. The assay was sufficiently sensitive to distinguish between antibody- and beta-chemokine-mediated fusion inhibition using serum samples from patient BX08, suggesting that the system may be useful for screening human sera for the presence of biologically significant antibodies.

Accessibility of nuclear DNA to triplex-forming oligonucleotides: the integrated HIV-1 provirus as a target.

The control of gene transcription by antigene oligonucleotides rests upon the specific recognition of double-helical DNA by triplex-forming oligonucleotides. The development of the antigene strategy requires access to the targeted DNA sequence within the chromatin structure of the cell nucleus. In this sudy we have used HIV-1 chronically infected cells containing the HIV provirus as endogenous genes to demonstrate that the integrated HIV-1 proviral genome is accessible to triplex-forming oligonucleotides within cell nuclei. An oligonucleotide-psoralen conjugate targeted to the polypurine tract (PPT) of the HIV-1 proviral sequence was used as a tool to convert the noncovalent triple-helical complex into a covalent lesion on genomic DNA after UV irradiation of cells. Triplex-derived adducts were analyzed using two different methods. The photo-induced psoralen cross-link prevented cleavage of the target sequence by DraI restriction endonuclease, and the sequence-specific inhibition of cleavage was revealed and quantitated by Southern blot analysis. A quantitative analysis of cross-linking efficiency was also carried out by a competitive PCR-based assay. These two approaches allowed us to demonstrate that a triplex-forming oligonucleotide can recognize and bind specifically to a 15-bp sequence within the chromatin structure of cell nuclei.

Human immunodeficiency virus type 1 Vif- mutant particles from restrictive cells: role of Vif in correct particle assembly and infectivity.

Disruption of the vif gene of human immunodeficiency virus (HIV) type 1 affects virus infectivity to various degrees, depending on the T-cell line used. We have concentrated our studies on true phenotypic Vif- mutant particles produced from CEMx174 or H9 cells. In a single round of infection, Vif- virus is approximately 25 (from CEMx174 cells) to 100 (from H9 cells) times less infectious than wild-type virus produced from these cells or than the Vif- mutant produced from HeLa cells. Vif- virions recovered from restrictive cells, but not from permissive cells, are abnormal both in terms of morphology and viral protein content. Notably, they contain much reduced quantities of envelope proteins and altered quantities of Gag and Pol proteins. Although wild-type and Vif- virions from restrictive cells contain similar quantities of viral RNA, no viral DNA synthesis was detectable after acute infection of target cells with phenotypically Vif- virions. To examine the possible role of Vif in viral entry, attempts were made to rescue the Vif- defect in H9 cells by pseudotyping Vif+ and Vif- HIV particles with amphotropic murine leukemia virus envelope. Vif- particles produced in the presence of HIV envelope could not be propagated when pseudotyped. In contrast, when only the murine leukemia virus envelope was present, significant propagation of Vif- HIV particles could be detected. These results demonstrate that Vif is required for proper assembly of the viral particle and for efficient HIV Env-mediated infection of target cells.

A highly defective HIV-1 group O provirus: evidence for the role of local sequence determinants in G-->A hypermutation during negative-strand viral DNA synthesis.

The sequence of 2350 nucleotides in the env and IN regions of a group O HIV-1 genome which is hypermutated throughout its entirety was compared to the equivalent sequence of a nonhypermutated genome from the same isolate. Almost 30% of G residues were affected by G-->A transitions. As previously reported, transitions occurred mainly at GpA and GpG dinucleotides, with a marked preference for changes of the 5'-proximal G residues in poly(G) stretches. Inspection of the sequences around the hypermutation sites revealed no bias when the mutation was at the 5' G residue of a GpG dinucleotide. In contrast, a preferred context for hypermutation at the 3' G (or at single G residues) could be defined. In addition to a preference for A residues immediately downstream of hypermutated 3' G residues, C residues were underrepresented in these positions. The observed context fits well with a model whereby G-->A mutation occurs by a combination of dislocation mutagenesis at GpA dinucleotides and direct misincorporation of dTTP at the 5' G of GpG dinucleotides. Furthermore, both runs of six G residues present in the polypurine tracts (PPTs) had escaped hypermutation, despite the fact that 95% of runs of three G residues contained at least one G-->A transition. This finding suggests that genomes with hypermutated PPT motifs had been selected against and provides direct evidence that hypermutation occurs during negative-strand DNA synthesis.

Isolation and envelope sequence of a highly divergent HIV-1 isolate: definition of a new HIV-1 group.

We report here the isolation and envelope sequence of a divergent HIV-1 isolate from a French woman with AIDS. This virus, HIV-1VAU, is closely related to the recently described Cameroonian viral isolates HIV-1ANT70 and HIV-1MVP5180, until now designated HIV-1 subtype O. Phylogenetic analysis reveals that the three viruses are equidistant from one another and that their mutual divergence is similar to what has been reported between the more conventional HIV-1 subtypes. Therefore, these three viruses could be included in a new viral group, HIV-1 group O (outgroup), distinct from the cluster of other HIV-1 isolates, which we will refer to as group M (Major group). The HIV-1 group O is currently emerging in western central Africa but its spread in Europe has already started.