Lentiviral gene therapy for X-linked chronic granulomatous disease


Chronic granulomatous disease (CGD) is a rare inherited disorder of phagocytic cells1,2. We report the initial results of nine severely affected X-linked CGD (X-CGD) patients who received ex vivo autologous CD34+ hematopoietic stem and progenitor cell-based lentiviral gene therapy following myeloablative conditioning in first-in-human studies (trial registry nos. NCT02234934 and NCT01855685). The primary objectives were to assess the safety and evaluate the efficacy and stability of biochemical and functional reconstitution in the progeny of engrafted cells at 12 months. The secondary objectives included the evaluation of augmented immunity against bacterial and fungal infection, as well as assessment of hematopoietic stem cell transduction and engraftment. Two enrolled patients died within 3 months of treatment from pre-existing comorbidities. At 12 months, six of the seven surviving patients demonstrated stable vector copy numbers (0.4–1.8 copies per neutrophil) and the persistence of 16–46% oxidase-positive neutrophils. There was no molecular evidence of either clonal dysregulation or transgene silencing. Surviving patients have had no new CGD-related infections, and six have been able to discontinue CGD-related antibiotic prophylaxis. The primary objective was met in six of the nine patients at 12 months follow-up, suggesting that autologous gene therapy is a promising approach for CGD patients.

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Fig. 1: Materials and methods (vector map and procedures).
Fig. 2: Biochemical and clinical evidence of successful engraftment.
Fig. 3: Analysis of vector integration site distributions and promotor methylation.

Data availability

All relevant data are included in the paper. Additional supporting data are available from the corresponding authors on request. All requests for raw and analyzed data and materials will be reviewed by the corresponding authors to verify whether the request is subject to any intellectual property or confidentiality obligations. Patient-related data not included in the paper were generated as part of clinical trials and may be subject to patient confidentiality.


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The authors thank the patients and families of those included in the study, and members of the Net4GCD consortium (https://www.net4CGD.eu) including: Eurofins Genomics Sequencing Europe, Germany (formerly GATC Biotech AG); F. Mavilio, Genethon, France (currently at the University of Modena, Italy, and at Audentes Therapeutics); J. Schwaeble, University Hospital Frankfurt, Germany; J. Reichenbach, University of Zurich, Switzerland; M. Cavazzana, APHP Necker, Paris, France; S. Blanche, APHP Necker, Paris, France; S. Pouillot, Genosafe, Evry, France (currently at Genethon); K. Kühlcke, Europäisches Institut für Forschung und Entwicklung von Transplantationstrategien GmbH (Eufets), Germany; and M. Schmidt, Deutsches Krebforschungszentrum, Heidelberg, Germany. Editorial support was paid for by Orchard Therapeutics, Boston, MA, USA. This work was supported by research grants from the California Institute of Regenerative Medicine (nos. CLIN2-08231 and FA1-00613-1), the Gene Therapy Resource Program from NHLBI, NIH (no. CRB-SSS-S-15-004351 1840), the NIAID Intramural Program, NET4CGD (FP7 EU grant agreement no. 305011) and the Wellcome Trust (no. 104807/Z/14/Z); by funding from the Department of Medicine (now Department of Pediatrics), Boston Children’s Hospital and the National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust/University College London Hospitals NHS Foundation Trust and University College London, and the French Muscular Dystrophy Association (AFM/Telethon). Clinical-grade vector was manufactured by Genethon BioProd/Yposeksi, Evry, France. Some oxidase activity analyses were funded in whole or in part by federal funds from the National Cancer Institute, NIH, under contract no. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the United States Government. Support was also provided by the Division of Intramural Research, NIAID, NIH.

Author information

The manuscript was written primarily by D.B. Kohn, A.J.T., D.A.W. and H.L.M. VISA analyses were performed by F.D.B., J.G., H.E.R. and J.K.E. M.G. and G.S. helped to construct the initial vector. G.H. and A.G. provided key resources for the conduct of studies, including clinical-grade vector, preclinical data package for CTA, study monitoring and pharmacovigilance and review of the manuscript. A.G. coordinated the Net4CGD consortium. C.B., E.M.K., S.-Y.P., K.L.S., M.A., K.F.B., U.C., S.S.d.R., M.J.D., C.Y.K., D.L.-R., C.R., N.I., K.G., K.S., J.X-B.D., J.D., E.C.M., D.T., L.D.W., C.A.B., T.P., D.B. Kuhns, L.B., P.E.N. and H.B.G. were involved in manufacture of the drug product, delivering the drug to patients for their respective clinics or performing analyses of patient samples. All authors had full access to all data, statistical reports and tables in the study and can take responsibility for the integrity of the data and accuracy of the data analysis. All authors approved the final manuscript.

Correspondence to Donald B. Kohn or Adrian J. Thrasher.

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Competing interests

D.B. Kohn, H.L.M., D.A.W. and A.J.T. are Scientific Advisory Board members, and H.B.G. is Chief Scientific Officer for Orchard Therapeutics. H.B.G. is an employee of and equity/stock holder in Orchard Therapeutics. A.J.T. is an equity/stock holder in Orchard Therapeutics. Orchard Therapeutics has obtained an exclusive option to license from Genethon for the rights and know-how related to the lentiviral vector G1XCGD. C.A.B. and T.P. consult for a sequencing service provider, and C.A.B. consults for Novimmune and SOBI. Eurofins Genomics Sequencing Europe (formerly GATC Biotech AG) is a for-profit company (sequencing service provider). The work performed by Eurofins Genomics Sequencing Europe included in the manuscript is provided to the greater scientific community as a fee-for-service product. E.C.M. reports Advisory Board attendance for Orchard Therapeutics. With regard to interests outside of the submitted work, L.D.W. reports grants from the St. Baldrick’s Foundation, Damon-Runyon Cancer Research Foundation and Alex’s Lemonade Stand Foundation, and personal fees from Magenta Therapeutics. A.J.T. reports Board membership and consultancy with Rocket Pharmaceuticals and Generation Bio, and Board membership with 4BIOCapital. S.-Y.P. reports salary support from Boston Children’s Hospital and a grant from NIH. K.L.S. reports personal fees and non-financial support from Orchard Therapeutics. E.M.K., G.S., M.A., K.F.B., U.C., S.S.d.R., M.J.D., C.Y.K., D.L.-R., C.R., N.I., K.G., K.S., J.D., E.C.M., D.B. Kuhns, J.G., H.R., J.K.E., G.H., P.E.N., F.D.B., M.G., J.X.-B.D. and A.G. declare no competing interests.

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Peer review information Kate Gao was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Schematic representation of the transfer plasmid pCCL_chimGP91_WPRE_Kana.

Cytomegalovirus (CMV) promoter; R, R region of long terminal repeat (LTR); U5, region unique to the 5’ LTR; Psi, encapsidation sequence; RRE, rev responsive element; cPPT, central polypurine tract; CTSG, cathepsin G 5’ minimal flanking regions; FES, human c-fes; coGP91, codon-optimized sequence CYBB gene which is translated into the cytochrome b-245, beta polypeptide; WPRE, mutated Woodchuck hepatitis virus post-transcriptional regulatory element; PPT, HIV-1 polyadenylated region (post-translational); dU3, region unique to the 3’ LTR (U3) of the HIV-1 LTR, in which a 400-bp deletion was introduced to inactivate the HIV-1 enhancer/promoter region; SV40 ori, Simian Virus 40 origin of replication; KANr, kanamycin resistance gene; COLE1, colicin E1 resistance gene.

Extended Data Fig. 2 Cell products and busulfan conditioning.

Panel a shows the final CD34+ cell products (left-axis) obtained for each patient. Panel b shows the VCN per infused cell post-transduction as determined by quantitative PCR. Transduction was also assessed by analyzing the percentage of vector CFUs PCR-positive for GX1CGD vector detected in the final cell product (Panel c). Vertical bars indicate mean and SD. Panel d displays the total net AUC busulfan exposure obtained by the sum of all pharmacokinetic measurements. Target total exposure of 70,000–75,000 ng ml-1 × h is indicated by the shaded area. AUC, area under the curve; CFU, colony-forming unit; PCR, polymerase chain reaction; SD, standard deviation; VCN, vector copy number.

Extended Data Fig. 3 Neutrophil DHR and VCN 24 months post treatment.

Panel a shows the mean (±SEM) of % DHR neutrophils up to 24 months post-treatment, as measured by a dihydrorhodamine oxidation assay. Panel b shows the mean neutrophil VCN (±SEM) up to 24 months post-treatment. DHR, dihydrorhodamine; SEM, standard error of mean; VCN, vector copy number.

Extended Data Fig. 4 X-CGD lineage VCN.

VCN determined by qPCR in multiple-cell lineages for patients surviving >1 month post-GT at each follow-up, showing stable integration of vector into multiple immune cell types. GT, gene therapy; qPCR, quantitative PCR; VCN, vector copy number.

Extended Data Fig. 5 Longitudinal analysis of inferred minimal population sizes.

Longitudinal analysis of minimal population size estimated using the CHAO1 method. The x axis shows time since cell infusion. D0 indicates the pre-infusion product. The y axis shows the numbers of unique integration sites (log scale). Cell types are color coded (bottom). For a few patients a reduced number of cell types were available for analysis. NK, natural killer; PBMC, peripheral blood mononuclear cell.

Extended Data Fig. 6 Catalogue of vector integration sites at genes of concern.

Catalogue of cell clones with integration sites within MECOM (MDS/EVI1), PRDM16, and SETBP2. These genes were chosen for analysis because they were targets of integration in expanded cell clones in the first γ-RV-based CGD trial, and were implicated in adverse outcomes. The x axis shows the time point queried, the y axis shows clonal abundance. Cell types queried are color coded (key at the bottom). No cell clones with integration sites near these genes of concern reaches ≥0.3% in abundance, and there is no evidence for longitudinal increases in proportion. CGD, chronic granulomatous disease; γ-RV, gammaretroviral; NK, natural killer; PBMC, peripheral blood mononuclear cell.

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Kohn, D.B., Booth, C., Kang, E.M. et al. Lentiviral gene therapy for X-linked chronic granulomatous disease. Nat Med 26, 200–206 (2020). https://doi.org/10.1038/s41591-019-0735-5

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