Persistent hematopoietic polyclonality after lentivirus-mediated gene therapy for Fabry disease

doi:10.1016/j.omtm.2023.01.003

. 2023 Jan 18:28:262-271.

doi: 10.1016/j.omtm.2023.01.003. eCollection 2023 Mar 9.

Persistent hematopoietic polyclonality after lentivirus-mediated gene therapy for Fabry disease

Amr H Saleh^{1

2}, Michael Rothe³, Dwayne L Barber^{1

4}, William M McKillop⁵, Graeme Fraser⁶, Chantal F Morel⁷, Axel Schambach^{3

8}, Christiane Auray-Blais⁹, Michael L West¹⁰, Aneal Khan¹¹, Daniel H Fowler¹², C Anthony Rupar^{13

14}, Ronan Foley¹⁵, Jeffrey A Medin^{5

16}, Armand Keating^{1

4

17}

Affiliations

¹ University Health Network, Toronto, ON, Canada.
² Department of Medicine, University of Alberta, Edmonton, AB, Canada.
³ Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
⁴ Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.
⁵ Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA.
⁶ Department of Oncology, McMaster University and Juravinski Hospital and Cancer Centre, Hamilton, ON, Canada.
⁷ Fred A. Litwin Family Centre in Genetic Medicine, Department of Medicine, University, Health Network, Toronto, ON, Canada.
⁸ Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.
⁹ Division of Medical Genetics, Department of Pediatrics, CIUSSS de l'Estrie-CHUS, Hospital Fleurimont, Université de Sherbrooke, Sherbrooke, QC, Canada.
¹⁰ Division of Nephrology, Department of Medicine, Dalhousie University, Halifax, NS, Canada.
¹¹ Department of Medical Genetics, Metabolics and Pediatrics, Alberta Children's Hospital, Cumming School of Medicine, Research Institute, University of Calgary, Calgary, AB, Canada.
¹² Rapa Therapeutics, Rockville, MD, USA.
¹³ Departments of Pathology and Laboratory Medicine and Pediatrics, Western University, London, ON, Canada.
¹⁴ Children's Health Research Institute, London, ON, Canada.
¹⁵ Department of Pathology and Molecular Medicine, McMaster University and Juravinski, Hospital and Cancer Centre, Hamilton, ON, Canada.
¹⁶ Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, USA.
¹⁷ Princess Margaret Cancer Centre, 610 University Avenue, 700U 6-325 Toronto, ON M5G 2M9, Canada.

PMID: 36816757
PMCID: PMC9932294
DOI: 10.1016/j.omtm.2023.01.003

Persistent hematopoietic polyclonality after lentivirus-mediated gene therapy for Fabry disease

Amr H Saleh et al. Mol Ther Methods Clin Dev. 2023.

. 2023 Jan 18:28:262-271.

doi: 10.1016/j.omtm.2023.01.003. eCollection 2023 Mar 9.

Authors

Affiliations

¹ University Health Network, Toronto, ON, Canada.
² Department of Medicine, University of Alberta, Edmonton, AB, Canada.
³ Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
⁴ Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.
⁵ Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA.
⁶ Department of Oncology, McMaster University and Juravinski Hospital and Cancer Centre, Hamilton, ON, Canada.
⁷ Fred A. Litwin Family Centre in Genetic Medicine, Department of Medicine, University, Health Network, Toronto, ON, Canada.
⁸ Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.
⁹ Division of Medical Genetics, Department of Pediatrics, CIUSSS de l'Estrie-CHUS, Hospital Fleurimont, Université de Sherbrooke, Sherbrooke, QC, Canada.
¹⁰ Division of Nephrology, Department of Medicine, Dalhousie University, Halifax, NS, Canada.
¹¹ Department of Medical Genetics, Metabolics and Pediatrics, Alberta Children's Hospital, Cumming School of Medicine, Research Institute, University of Calgary, Calgary, AB, Canada.
¹² Rapa Therapeutics, Rockville, MD, USA.
¹³ Departments of Pathology and Laboratory Medicine and Pediatrics, Western University, London, ON, Canada.
¹⁴ Children's Health Research Institute, London, ON, Canada.
¹⁵ Department of Pathology and Molecular Medicine, McMaster University and Juravinski, Hospital and Cancer Centre, Hamilton, ON, Canada.
¹⁶ Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, USA.
¹⁷ Princess Margaret Cancer Centre, 610 University Avenue, 700U 6-325 Toronto, ON M5G 2M9, Canada.

PMID: 36816757
PMCID: PMC9932294
DOI: 10.1016/j.omtm.2023.01.003

Abstract

The safety and efficacy of lentivirus-mediated gene therapy was recently demonstrated in five male patients with Fabry disease-a rare X-linked lysosomal storage disorder caused by GLA gene mutations that result in multiple end-organ complications. To evaluate the risks of clonal dominance and leukemogenesis, which have been reported in multiple gene therapy trials, we conducted a comprehensive DNA insertion site analysis of peripheral blood samples from the five patients in our gene therapy trial. We found that patients had a polyclonal integration site spectrum and did not find evidence of a dominant clone in any patient. Although we identified vector integrations near proto-oncogenes, these had low percentages of contributions to the overall pool of integrations and did not persist over time. Overall, we show that our trial of lentivirus-mediated gene therapy for Fabry disease did not lead to hematopoietic clonal dominance and likely did not elevate the risk of leukemogenic transformation.

Keywords: Fabry disease; clinical trial; gene therapy; lentiviral integration; lentiviral safety.

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Conflict of interest statement

D.L.B. was partially paid from a sponsored research agreement from AVROBIO, Inc. C.A.-B. has received a service contract and honoraria for biomarker analysis with AVROBIO, Inc. and a grant from CIHR. C.F.M. has received grants, personal fees, and non-financial support from Takeda Pharmaceuticals (previously Shire HGT); grants, personal fees, and non-financial support from Sanofi-Genzyme; and non-financial support from Amicus Therapeutics. A. Khan received grants, consulting fees, revenue distribution agreement, speaker fees, and travel support with AVROBIO, Inc., as well as has a revenue distribution agreement with University Health Network regarding gene therapy using technology from this work. M.L.W. has received research grants, consulting fees, speaker fees, and travel support from Amicus Therapeutics, Protalix, Sanofi-Genzyme, and Takeda and has a revenue distribution agreement with University Health Network regarding gene therapy using technology from this work. C.A.R. has the following financial relationships to disclose: the Biochemical Genetics clinical diagnostic laboratory at his home institution is contracted by AVROBIO, Inc., to assay enzymes on a fee-for--service basis. He is the laboratory director but receives no personal compensation. J.A.M. is on the scientific advisory board of Rapa Therapeutics; has received honoraria from Sanofi Genzyme and Shire; is a co-founder and shareholder of AVROBIO, Inc.; and has received grants from Canadian Institutes of Health Research and Kidney Foundation of Canada and AVROBIO, Inc. A. Keating has received a consultancy fee from AVROBIO, Inc., unrelated to this study. A.S. and M.R. have a service contract and received consultancy fees from AVROBIO, Inc., unrelated to this study.

Figures

**Figure 1**
Unique integration sites at 6 months correlate with the number of infused CD34⁺ cells/kg We observe a trend regarding the number of unique integration sites (UISs) and the number of CD34⁺ cells infused in each patient, plotted as a function of CD34⁺ cells/kilogram. Linear regression was performed, and the equation, non-parametric correlation (Spearman) co-efficient, and p value are illustrated.

**Figure 2**
Top 10 integration sites The top 10 integration sites of control samples (A) and patients 1–5 at the different time points (B) are shown here. Each colored bar represents a separate insertion site, and the gray bars represent all other insertion sites. Below each bar, the gene symbols closest to the integration sites are listed. ∗, integration was within a transcription unit. ∼, insertion was within 50 kb of a cancer-related gene. UIS, unique integration sites.

**Figure 3**
Tendency for vector integration in genes and close to proto-oncogenes compared with matched random control sites We found a higher tendency for vector integrations inside transcriptional units (A) and within 100 kb of proto-oncogenes (B) compared with matched random control (mrc) sites). ∗p_Mann-Whitney < 0.05, ∗∗∗p_Mann-Whitney < 0.001.

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[1] Zarate Y.A., Hopkin R.J. Fabry's disease. Lancet. 2008;372:1427–1435. - PubMed

[2] Zarate Y.A., Hopkin R.J. Fabry's disease. Lancet. 2008;372:1427–1435. - PubMed

[3] Ortiz A., Germain D.P., Desnick R.J., Politei J., Mauer M., Burlina A., Eng C., Hopkin R.J., Laney D., Linhart A., et al. Fabry disease revisited: management and treatment recommendations for adult patients. Mol. Genet. Metab. 2018;123:416–427. - PubMed

[4] Ortiz A., Germain D.P., Desnick R.J., Politei J., Mauer M., Burlina A., Eng C., Hopkin R.J., Laney D., Linhart A., et al. Fabry disease revisited: management and treatment recommendations for adult patients. Mol. Genet. Metab. 2018;123:416–427. - PubMed

[5] Khan A., Barber D.L., Huang J., Rupar C.A., Rip J.W., Auray-Blais C., Boutin M., O'Hoski P., Gargulak K., McKillop W.M., et al. Lentivirus-mediated gene therapy for Fabry disease. Nat. Commun. 2021;12:1178. - PMC - PubMed

[6] Khan A., Barber D.L., Huang J., Rupar C.A., Rip J.W., Auray-Blais C., Boutin M., O'Hoski P., Gargulak K., McKillop W.M., et al. Lentivirus-mediated gene therapy for Fabry disease. Nat. Commun. 2021;12:1178. - PMC - PubMed

[7] Hacein-Bey-Abina S., Von Kalle C., Schmidt M., McCormack M.P., Wulffraat N., Leboulch P., Lim A., Osborne C.S., Pawliuk R., Morillon E., et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science. 2003;302:415–419. - PubMed

[8] Hacein-Bey-Abina S., Von Kalle C., Schmidt M., McCormack M.P., Wulffraat N., Leboulch P., Lim A., Osborne C.S., Pawliuk R., Morillon E., et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science. 2003;302:415–419. - PubMed

[9] Hacein-Bey-Abina S., Garrigue A., Wang G.P., Soulier J., Lim A., Morillon E., Clappier E., Caccavelli L., Delabesse E., Beldjord K., et al. Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. J. Clin. Invest. 2008;118:3132–3142. - PMC - PubMed

[10] Hacein-Bey-Abina S., Garrigue A., Wang G.P., Soulier J., Lim A., Morillon E., Clappier E., Caccavelli L., Delabesse E., Beldjord K., et al. Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. J. Clin. Invest. 2008;118:3132–3142. - PMC - PubMed

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Persistent hematopoietic polyclonality after lentivirus-mediated gene therapy for Fabry disease

Affiliations

Persistent hematopoietic polyclonality after lentivirus-mediated gene therapy for Fabry disease

Authors

Affiliations

Abstract

Conflict of interest statement

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Abstract

Conflict of interest statement

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