BACKGROUND:Renizgamglogene autogedtemcel (reni-cel) is an investigational clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a gene-edited autologous hematopoietic stem-cell therapy. The therapy was designed to disrupt the BCL11A binding sites in the HBG1 and HBG2 promoters to reactivate fetal hemoglobin production for the treatment of sickle cell disease. METHODS:We conducted a phase 1-2, multicenter, open-label, single-group study involving patients with severe sickle cell disease who were 12 to 50 years of age and had had at least two severe vaso-occlusive events per year in the previous 2 years. The patients received a single infusion of reni-cel after myeloablative conditioning with busulfan. The patients were monitored for engraftment, hemoglobin-related measures, allelic editing levels, vaso-occlusive events, and adverse events over a 24-month period. The study was terminated early on the basis of the sponsor's reassessment of clinical development priorities. Results of an analysis that was not prespecified are reported. RESULTS:As of October 29, 2024, a total of 28 patients with severe sickle cell disease had been treated with reni-cel. The median duration of follow-up was 9.5 months (range, 0.7 to 25.2). Among 27 patients who had neutrophil and platelet engraftment by the data-cutoff date, neutrophil engraftment occurred after a median of 23 days (range, 14 to 29), and platelet engraftment occurred after a median of 25 days (range, 17 to 51). At month 6, among 18 patients with at least 6 months of available data, the mean (±SD) total hemoglobin level (9.8±1.7 g per deciliter at baseline) had increased to 13.8±1.9 g per deciliter, and the mean percentage of fetal hemoglobin (2.5±2.5% at baseline) had increased to 48.1±3.2%; both measures were maintained at or above these values thereafter. One patient had two severe vaso-occlusive events after infusion. Adverse events were consistent with those that occur after myeloablative busulfan-based conditioning and autologous hematopoietic stem-cell transplantation. CONCLUSIONS:Treatment with reni-cel led to normalization of the total hemoglobin level and an increase in the percentage of fetal hemoglobin, with no vaso-occlusive events occurring in 27 of 28 patients after infusion. These results support further investigation of this gene-editing approach in the treatment of severe sickle cell disease. (Funded by Editas Medicine; RUBY ClinicalTrials.gov number, NCT04853576.).

BACKGROUND:Renizgamglogene autogedtemcel (reni-cel) is an investigational clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a gene-edited autologous hematopoietic stem-cell therapy. The therapy was designed to disrupt the BCL11A binding sites in the HBG1 and HBG2 promoters to reactivate fetal hemoglobin production for the treatment of transfusion-dependent β-thalassemia. METHODS:We conducted a phase 1-2, multicenter, open-label, single-group study of reni-cel in participants 18 to 35 years of age with transfusion-dependent β-thalassemia. The participants received myeloablative conditioning with busulfan before reni-cel infusion. The primary end points were neutrophil engraftment by 42 days after infusion and frequency and severity of adverse events. Participants were monitored for hemoglobin-related measures and transfusion independence. The study was terminated early on the basis of the sponsor's reassessment of clinical development priorities. Results of an analysis that was not prespecified are reported. RESULTS:Nine participants with transfusion-dependent β-thalassemia (four β0/β0 or β0/β0-like and five non-β0/β0 genotypes) received reni-cel and were included in the analysis. The median duration of postinfusion follow-up was 17.5 months (range, 3.8 to 23.4), and six participants could be evaluated for transfusion independence at 12 months or more. All the participants had neutrophil and platelet engraftment by 42 days after infusion. Rapid increases in total and fetal hemoglobin levels resulted in each of the nine participants being transfusion-free at their last follow-up visit. The six participants who could be evaluated at 12 months or later were transfusion-independent. The mean total and fetal hemoglobin levels were greater than 12 g per deciliter and greater than 11 g per deciliter, respectively, between months 6 and 18. A total of 69 grade 3 or 4 adverse events with onset or worsening during or after reni-cel infusion were reported in the nine participants. Six serious adverse events (infections, pyrexia, or pneumonitis) were reported in four participants. Adverse events were generally consistent with myeloablative conditioning. One patient had decreased lymphocyte counts attributed to reni-cel. CONCLUSIONS:Treatment with reni-cel resulted in rapid neutrophil engraftment, an increase in fetal hemoglobin expression, and transfusion independence. These data support further investigation of Cas12a gene editing of the promoters of HBG1 and HBG2 in the treatment of transfusion-dependent β-thalassemia. (Funded by Editas Medicine; EdiThal ClinicalTrials.gov number, NCT05444894.).

Background CEP290-associated inherited retinal degeneration causes severe early-onset vision loss due to pathogenic variants in CEP290. EDIT-101 is a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing complex designed to treat inherited retinal degeneration caused by a specific damaging variant in intron 26 of CEP290 (IVS26 variant). Methods We performed a phase 1-2, open-label, single-ascending-dose study in which persons 3 years of age or older with CEP290-associated inherited retinal degeneration caused by a homozygous or compound heterozygous IVS26 variant received a subretinal injection of EDIT-101 in the worse (study) eye. The primary outcome was safety, which included adverse events and dose-limiting toxic effects. Key secondary efficacy outcomes were the change from baseline in the best corrected visual acuity, the retinal sensitivity detected with the use of full-field stimulus testing (FST), the score on the Ora-Visual Navigation Challenge mobility test, and the vision-related quality-of-life score on the National Eye Institute Visual Function Questionnaire-25 (in adults) or the Children's Visual Function Questionnaire (in children). Results EDIT-101 was injected in 12 adults 17 to 63 years of age (median, 37 years) at a low dose (in 2 participants), an intermediate dose (in 5), or a high dose (in 5) and in 2 children 9 and 14 years of age at the intermediate dose. At baseline, the median best corrected visual acuity in the study eye was 2.4 log(10) of the minimum angle of resolution (range, 3.9 to 0.6). No serious adverse events related to the treatment or procedure and no dose-limiting toxic effects were recorded. Six participants had a meaningful improvement from baseline in cone-mediated vision as assessed with the use of FST, of whom 5 had improvement in at least one other key secondary outcome. Nine participants (64%) had a meaningful improvement from baseline in the best corrected visual acuity, the sensitivity to red light as measured with FST, or the score on the mobility test. Six participants had a meaningful improvement from baseline in the vision-related quality-of-life score. Conclusions The safety profile and improvements in photoreceptor function after EDIT-101 treatment in this small phase 1-2 study support further research of in vivo CRISPR-Cas9 gene editing to treat inherited retinal degenerations due to the IVS26 variant of CEP290 and other genetic causes.

Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an essential gene and designed a cargo template so that correct knock-in would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive insertions and deletions would undergo negative selection. This technology, called SLEEK (SeLection by Essential-gene Exon Knock-in), achieved knock-in efficiencies of more than 90% in clinically relevant cell types without impacting long-term viability or expansion. SLEEK knock-in rates in T cells are more efficient than state-of-the-art TRAC knock-in with AAV6 and surpass more than 90% efficiency even with non-viral DNA cargos. As a clinical application, natural killer cells generated from induced pluripotent stem cells containing SLEEK knock-in of CD16 and mbIL-15 show substantially improved tumor killing and persistence in vivo.

Background: EDIT-301, an investigational gene-edited autologous hematopoietic stem cell medicine, has a unique genomic modification at the γ-globin gene (HBG1/HBG2) promoters where multiple naturally occurring mutations for hereditary persistence of fetal hemoglobin (HPFH) reside to reactivate γ-globin expression and increase HbF production. EDIT-301 is manufactured using a highly efficient and specific, proprietary AsCas12a. In preclinical studies, editing of this region in CD34+ cells from patients with SCD led to ≥80% editing, robust HbF production, no off-target editing, and significantly reduced sickling of EDIT-301-derived erythroid progeny. Aims: The RUBY trial (NCT04853576), a Phase I/II, multicenter, open-label, single-arm study, evaluates the safety, tolerability, and efficacy of EDIT-301 in subjects with severe SCD. Four subjects have received EDIT-301 treatment. Preliminary clinical data on gene editing, safety, and efficacy are reported. Methods: Subjects 18–50 years old must have a diagnosis of severe SCD defined as ≥2 severe vaso-occlusive events (VOEs) per year in the 2-year period prior to informed consent. Autologous CD34+ hematopoietic stem and progenitor cells are collected by apheresis after plerixafor mobilization and edited at the HBG1/HBG2 promoter with AsCas12a. After myeloablative conditioning with busulfan, subjects received a single infusion of EDIT-301 (≥3 × 106 CD34+ cells/kg), and were monitored for engraftment, total hemoglobin (Hb), HbF production, mean HbF concentration/F-cell (MCH-F/F-cell), percentage of F-cells, markers of hemolysis, transfusion requirement, VOEs, and adverse events (AEs) for 24-months. Results: Editing of CD34+ cells using AsCas12a resulted in ≥80% editing in study participants’ cells (N=8). As of March 1, 2023, Subjects 1 and 2 are 8- and 4-months post-EDIT-301 infusion, respectively, and Subjects 3 and 4 are <1-month post-EDIT-301 infusion. Neutrophil and platelet engraftment were achieved within 23 and 19 days (Subject 1) and within 29 and 37 days (Subject 2) of EDIT-301 infusion, respectively. At last data points available, Subjects 1 and 2 had normal Hb concentrations, HbF levels >35% (Figure 1), MCH-F/F-cell >10.0 pg/F-cell, and no reported VOEs. Hb increased by 4.5 g/dL from baseline (BL) to 16.4 g/dL at 6 months (Subject 1) and increased by 3.6 g/dL from BL to 12.1 g/dL at 3 months post-EDIT-301 infusion (Subject 2). Percentage of F-cells was 96.5% at 6 months (Subject 1). All markers of hemolysis improved or normalized. Editing levels in peripheral blood nucleated cells were >80% in both subjects. The safety profile of EDIT-301 was consistent with myeloablative conditioning with busulfan, with no reported EDIT-301-related AEs and no serious adverse events (SAEs) after EDIT-301 infusion. Summary/Conclusion: These preliminary data demonstrate successful engraftment, a rapid and sustained increase in total Hb, HbF level, and percentage of F-cells, improvements in key markers of hemolysis, and a favorable safety profile in subjects treated with EDIT-301. These preliminary data demonstrated clinical proof of concept and are promising for the first clinical use of AsCas12a-based gene editing of the globin gene (HBG1/HBG2) promoters, thereby supporting further investigation of EDIT-301 in the RUBY clinical trial. Updated data will be presented.Keywords: Globin gene, Sickle cell disease, Gene therapy

Vacuolar-type ATPases (V-ATPases) are rotary enzymes that acidify intracellular compartments in eukaryotic cells. These multi-subunit complexes consist of a cytoplasmic V1 region that hydrolyzes ATP and a membrane-embedded VO region that transports protons. V-ATPase activity is regulated by reversible dissociation of the two regions, with the isolated V1 and VO complexes becoming autoinhibited on disassembly and subunit C subsequently detaching from V1. In yeast, assembly of the V1 and VO regions is mediated by the regulator of the ATPase of vacuoles and endosomes (RAVE) complex through an unknown mechanism. We used cryogenic-electron microscopy of yeast V-ATPase to determine structures of the intact enzyme, the dissociated but complete V1 complex and the V1 complex lacking subunit C. On separation, V1 undergoes a dramatic conformational rearrangement, with its rotational state becoming incompatible for reassembly with VO. Loss of subunit C allows V1 to match the rotational state of VO, suggesting how RAVE could reassemble V1 and VO by recruiting subunit C. V-ATPases acidify the intracellular compartments of eukaryotic cells and their activity is regulated by reversible dissociation of the complex. Cryo-EM structures show the conformational changes associated with assembly and autoinhibition of V-ATPase.

Mutations in the human Rogdi protein cause Kohlschutter-Tonz syndrome, which is characterized by early-onset seizures, developmental defects, and defective deposition of tooth enamel (amelogenesis imperfecta). The cellular function of Rogdi is not known. The yeast RAVE (regulator of ATPase of vacuoles and endosomes) complex and mammalian Rabconnectin-3 complexes catalyze assembly of certain subpopulations of the V-ATPase proton pump and thus help to regulate pH homeostasis. The yeast RAVE complex is composed of three subunits: Rav1, Rav2, and Skp1. Skp1 is a multifunctional scaffold protein, but both Rav1 and Rav2 act specifically in V-ATPase assembly. Rabconnectin-3 complexes have been reported to contain two larger subunits, Rbcn3α and 3β; both subunits are predicted to have structural similarity to Rav1. No mammalian homologue of yeast Rav2 had been identified. However, despite limited sequence homology, we find that the yeast Rav2 sequence can be modeled with very high confidence on a recent structure of human Rogdi (H. Lee et al. (2017) Sci. Rep. 7:3972). Expression of human Rogdi in yeast complements the V-ATPase-associated growth defects of a yeast rav2∆ mutant. Complementation requires the presence of Rav1, suggesting that Rogdi acts as part of the RAVE complex rather than bypassing RAVE function. Consistent with this, Rogdi copurifies with a FLAG-tagged Rav1 protein. Yeast Rav2 binds to the N-terminal β-propeller region of Rav1. Two-hybrid assays indicate that Rogdi can interact with the same region of Rav1, as well as binding to the N-terminal β-propeller regions of Rbcn3α and 3β isoforms. Based on these data, we hypothesize that Rogdi is a subunit of mammalian Rabconnectin-3 complexes. WDR72 is a possible Rbcn3β isoform in humans. Mutations in the N-terminal β-propeller domain of WDR72 also cause amelogenesis imperfecta, as well as distal renal tubule acidosis, a disease associated with defects in V-ATPase activity. We are testing whether these mutations affect the interaction between Rogdi and WDR72, potentially linking Rogdi with both Rabconnectin-3 and V-ATPase function.

The yeast RAVE (Regulator of H+-ATPase of Vacuolar and Endosomal membranes) complex and Rabconnectin-3 complexes of higher eukaryotes regulate acidification of organelles such as lysosomes and endosomes by catalyzing V-ATPase assembly. V-ATPases are highly conserved proton pumps consisting of a peripheral V-1 subcomplex that contains the sites of ATP hydrolysis, attached to an integral membrane V-o subcomplex that forms the transmembrane proton pore. Reversible disassembly of the V-ATPase is a conserved regulatory mechanism that occurs in response to multiple signals, serving to tune ATPase activity and compartment acidification to changing extracellular conditions. Signals such as glucose deprivation can induce release of V-1 from V-o, which inhibits both ATPase activity and proton transport. Reassembly of V-1 with V-o restores ATP-driven proton transport, but requires assistance of the RAVE or Rabconnectin-3 complexes. Glucose deprivation triggers V-ATPase disassembly in yeast and is accompanied by binding of RAVE to V-1 subcomplexes. Upon glucose readdition, RAVE catalyzes both recruitment of V-1 to the vacuolar membrane and its reassembly with V-o. The RAVE complex can be recruited to the vacuolar membrane by glucose in the absence of V-1 subunits, indicating that the interaction between RAVE and the V-o membrane domain is glucose-sensitive. Yeast RAVE complexes also distinguish between organelle-specific isoforms of the V-o a-subunit and thus regulate distinct V-ATPase subpopulations. Rabconnectin-3 complexes in higher eukaryotes appear to be functionally equivalent to yeast RAVE. Originally isolated as a two-subunit complex from rat brain, the Rabconnectin-3 complex has regions of homology with yeast RAVE and was shown to interact with V-ATPase subunits and promote endosomal acidification. Current understanding of the structure and function of RAVE and Rabconnectin-3 complexes, their interactions with the V-ATPase, their role in signal-dependent modulation of organelle acidification, and their impact on downstream pathways will be discussed.

The vacuolar H+-ATPase (V-ATPase) is a highly conserved proton pump responsible for the acidification of intracellular organelles in virtually all eukaryotic cells. V-ATPases are regulated by the rapid and reversible disassembly of the peripheral V-1 domain from the integral membrane V-o domain, accompanied by release of the V-1 C subunit from both domains. Efficient reassembly of V-ATPases requires the Regulator of the H+-ATPase of Vacuoles and Endosomes (RAVE) complex in yeast. Although a number of pairwise interactions between RAVE and V-ATPase subunits have been mapped, the low endogenous levels of the RAVE complex and lethality of constitutive RAV1 overexpression have hindered biochemical characterization of the intact RAVE complex. We describe a novel inducible overexpression system that allows purification of native RAVE and RAVE-V1 complexes. Both purified RAVE and RAVE-V1 contain substoichiometric levels of subunit C. RAVE-V1 binds tightly to expressed subunit C in vitro, but binding of subunit C to RAVE alone is weak. Neither RAVE nor RAVE-V1 interacts with the N-terminal domain of V-o subunit Vph1 in vitro. RAVE-V1 complexes, like isolated V-1, have no MgATPase activity, suggesting that RAVE cannot reverse V1 inhibition generated by rotation of subunit H and entrapment of MgADP that occur upon disassembly. However, purified RAVE can accelerate reassembly of V-1 carrying a mutant subunit H incapable of inhibition with V-o complexes reconstituted into lipid nanodiscs, consistent with its catalytic activity in vivo. These results provide new insights into the possible order of events in V-ATPase reassembly and the roles of the RAVE complex in each event.

V‐ATPases are highly conserved proton pumps that acidify multiple organelles, including lysosomes, endosomes, the late Golgi apparatus, synaptic vesicles, and other regulated secretory granules. Precise tuning of the luminal pH of these organelles is critical for function, but the factors governing organelle pH control are not completely understood. V‐ATPases are multisubunit complexes consisting of a peripheral subcomplex (V1) that contains sites for ATP hydrolysis and an integral membrane subcomplex (Vo) that contains the proton pore. The Vo a‐subunit is the largest subunit. It is comprised of an N‐terminal cytosolic domain and a C‐terminal domain that forms part of the proton pore. Most organisms encode multiple isoforms of the Vo a‐subunit that exhibit organelle‐specific localization. We hypothesize that the N‐terminal (NT) domains of a‐subunit isoforms participate in distinct cellular interactions that are critical for isoform‐specific V‐ATPase localization, activity, and regulation. In yeast cells, there are two organelle‐specific isoforms of the Vo a‐subunit, Vph1 and Stv1. Vph1‐containing V‐ATPases transit through the secretory pathway en route to the lysosome‐like vacuole. Their activity is regulated by reversible disassembly in response to glucose levels, and they require interaction with the yeast RAVE (regulator of acidification of vacuoles and endosomes) for both their initial biosynthetic assembly and for glucose‐dependent reassembly. The Vph1NT domain binds directly to RAVE and is responsible for glucose‐sensitive interactions with the RAVE complex. In addition, the vacuolar lipid PI(3,5)P2 promotes assembly and activity of Vph1‐containing V‐ATPases. Mutations in Vph1NT compromise PI(3,5)P2‐induced activation, suggesting direct binding to lipid. In contrast, Stv1‐containing V‐ATPases assemble and function in the absence of the RAVE complex, and Stv1NT does not bind to RAVE. However, Stv1NT binds to the Golgi‐enriched lipid PI(4)P, and mutations that abolish PI(4)P binding compromise Golgi retention of Stv1‐containing V‐ATPases. The aNT domains of the four human Vo a‐subunit isoforms exhibit differential recognition of phosphoinositide lipids. Some human aNT domains interact with the human homologue of the RAVE complex. These data indicate that NT domains of Vo a‐subunit isoforms encode information for localization and regulation of V‐ATPase activity that help determine organelle pH and protect cells from stress.Support or Funding InformationNIH R01 GM127364 and NIH R01 GM126020

The yeast vacuolar H+-ATPase (V-ATPase) of budding yeast (Saccharomyces cerevisiae) is regulated by reversible disassembly. Disassembly inhibits V-ATPase activity under low-glucose conditions by releasing peripheral V1 subcomplexes from membrane-bound Vo subcomplexes. V-ATPase reassembly and reactivation requires intervention of the conserved regulator of H+-ATPase of vacuoles and endosomes (RAVE) complex, which binds to cytosolic V1 subcomplexes and assists reassembly with integral membrane Vo complexes. Consistent with its role, the RAVE complex itself is reversibly recruited to the vacuolar membrane by glucose, but the requirements for its recruitment are not understood. We demonstrate here that RAVE recruitment to the membrane does not require an interaction with V1. Glucose-dependent RAVE localization to the vacuolar membrane required only intact Vo complexes containing the Vph1 subunit, suggesting that the RAVE-Vo interaction is glucose-dependent. We identified a short conserved sequence in the center of the RAVE subunit Rav1 that is essential for the interaction with Vph1 in vivo and in vitro. Mutations in this region resulted in the temperature- and pH-dependent growth phenotype characteristic of ravΔ mutants. However, this region did not account for glucose sensitivity of the Rav1-Vph1 interaction. We quantitated glucose-dependent localization of a GFP-tagged RAVE subunit to the vacuolar membrane in several mutants previously implicated in altering V-ATPase assembly state or glucose-induced assembly. RAVE localization did not correlate with V-ATPase assembly levels reported previously in these mutants, highlighting both the catalytic nature of RAVE's role in V-ATPase assembly and the likelihood of glucose signaling to RAVE independently of V1.

Lysosomes and vacuoles are the most acidic organelles in eukaryotic cells and play a central role in cellular metabolism – serving as a location that coordinates anabolic and catabolic processes. This coordination requires the activity of a highly conserved proton pump, the Vacuolar H+‐ATPase (V‐ATPase). Interestingly, both V‐ATPase activity and a cell's metabolic state are dependent on glucose levels. Glucose deprivation induces the V‐ATPase to disassemble into 3 distinct subcomplexes: V1, V1C, and Vo. V1 and V1C are released from Vo at the vacuolar membrane, and this disassembly silences both ATP hydrolysis in V1 and proton transport through Vo. Reassembly occurs rapidly but requires both glucose readdition and a conserved V‐ATPase‐specific assembly factor known as the RAVE (Regulator of the H+‐ATPase of Vacuolar and Endosomes) complex in yeast. The RAVE complex, composed of Rav1, Rav2, and Skp1, interacts with each V‐ATPase subcomplex and, upon glucose readdition, recruits cytosolic V1 and V1C to Vo at the vacuolar membrane to allow V‐ATPase reassembly (Smardon et al., (2015) J. Biol. Chem 290:27511). Although RAVE is essential for efficient V‐ATPase reassembly, how RAVE targets the vacuolar membrane in a glucose‐dependent manner and promotes V‐ATPase reassembly is not understood.Like the V1 subcomplex and V1C subunit, the RAVE complex is reversibly recruited to the vacuolar membrane in response to glucose. Interestingly, we found that RAVE requires the presence of Vo, but neither V1 nor V1C, for its glucose‐dependent vacuolar localization. We identified a 6‐amino acid, conserved motif within Rav1 that is essential for RAVE's vacuolar localization in vivo. In vitro, deletion of this motif diminishes binding between Rav1 and the cytosolic N‐terminal domain of Vo subunit Vph1. These data suggest that this motif is essential for RAVE to identify Vo subunit Vph1 at the vacuolar membrane, but they do not explain the release of RAVE from the membrane upon glucose deprivation. We seek to determine the signaling mechanism and structural changes involved in RAVE's glucose‐dependent activities. V‐ATPase activity, glycolytic enzymes, the Ras/cAMP pathway, PI(3,5)P2 levels, and cytoskeletal elements have all been implicated in V‐ATPase assembly. We are genetically or chemically silencing the activity of each of these factors and assessing the glucose‐dependent localization of GFP‐tagged RAVE subunits. Initial results indicate that RAVE cycles on and off the vacuolar membrane even in the presence of an assembled but inactive V‐ATPase mutant that is incapable of disassembly. Similar experiments will determine the effects of other factors. Detailed biochemical characterization of the RAVE complex has been thwarted by low expression levels of RAVE subunits. However, we can now express and purify milligram quantities of the RAVE complex alone or bound to V1. This will allow us to identify glucose‐sensitive interactions with RAVE in vitro and to test the hypothesis that RAVE undergoes a glucose‐sensitive conformation change that reversibly exposes the Rav1‐Vph1 binding site. This work addresses the molecular mechanisms governing RAVE‐mediated V‐ATPase reassembly and is essential to understanding the central role of the V‐ATPase in cellular metabolism.Support or Funding InformationNIH GM127364This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Artemisia vulgaris L. was collected from various regions in the USA and Montreal Canada. Gas Chromatography-Mass Spectrometry was used to identify the analytes present in the volatiles extracted by headspace solid-phase microextraction of the crushed leaves and flowers. Four distinct chemotypes are were found: One featuring the coexistence of ar-curcumene and α-zingiberene; two marked by the presence or absence of thujone and santolinatriene; and a fourth characterized by the presence of crysanthenyl acetate (40%). DNA was used to confirm the identity of Artemisia vulgaris L.