Whole Exome Sequencing of Patients Treated with Adeno-associated Virus Serotype 8-Factor IX (AAV8-FIX) Gene Therapy Reveals Potential Determinants of Persistent Transgene Expression
ISTH Academy. reipert b. 07/08/19; 273987; OC 31.1 Topic: Hemophilia Gene Therapy
birgit reipert
birgit reipert
Login now to access Regular content available to all registered users.

Access to Premium content is currently a membership benefit.

Click here to join ISTH or renew your membership.

You may also access ISTH content "anytime, anywhere" with the FREE ISTH Academy App for iOS and Android.
Abstract
Discussion Forum (0)
Rate & Comment (0)

OC 31.1

Whole Exome Sequencing of Patients Treated with Adeno-associated Virus Serotype 8-Factor IX (AAV8-FIX) Gene Therapy Reveals Potential Determinants of Persistent Transgene Expression

I. Bilic1, P. Monahan2, V. Berg3, F. Scheiflinger1, B.M. Reipert1
1Baxalta Innovations GmbH, a Takeda Company, Vienna, Austria, 2Spark Therapeutics, Philadelphia, United States, 3IMC University of Applied Sciences Krems, Krems, Austria

Main Topic: Hemophilia and Bleeding (including Transfusion)
Category: Hemophilia Gene Therapy

Background: The clinical phase I/II study investigated BAX335, an AAV8-based gene therapy for FIX replacement in patients with hemophilia B. BAX335 was well-tolerated in all patients with no clinical thrombosis and no evidence of inhibitors or other FIX-directed immunity. Persistent therapeutic FIX activity of 20% without bleeding or replacement therapy was achieved in only one patient for >4 years (Index patient). In all other patients, FIX activity declined after 5-11 weeks and could not be rescued by corticosteroid treatment.
Aims: Identification of genetic predispositions as molecular determinants for long-term transgene expression in the Index patient using whole exome sequencing (WES).
Methods: High-coverage WES of genomic DNA from patients treated with BAX335 was combined with state-of-the-art variant identification and prioritization. Index patient was compared to the remaining seven patients to identify variants potentially mediating persistent transgene expression. Prioritized variants were evaluated according to the predicted deleteriousness using CADD, PolyPhen-2 and SIFT algorithms and to the phenotypic manifestations of their target genes.
Results: Two of the identified variants found in the index patient could impact transgene expression. The first are compound heterozygous variants of the nuclear receptor co-repressor complex SMRT/NCOR2, which mediates transcriptional silencing by tethering general chromatin-modifying enzymes to target genes. Since chromatinized episomal AAV-transgene is regulated by histone modifications1,2, the identified SMRT variants highlight protection from heterochromatinization as one of the mechanisms for ensuring persistent transgene expression.
The most indicative variant was a heterozygous missense mutation in the interleukin-6 receptor gene (IL-6R). IL-6R haploinsufficiency is well documented3-5, thus the identified variant might decrease sensitivity to IL-6-mediated inflammatory stress caused by BAX335 (AAV8-capsid and/or transgene) and protect targeted hepatocytes.
Conclusions: Two important parameters required for successful liver-targeting gene therapy are implicated: (i) design of robust expression cassette resistant to gene silencing by heterochromatinization and (ii) protection of targeted hepatocytes from inflammatory stress induced by viral load.


Ref. Nr. Reference
1. Penaud-Budloo M, Le Guiner C, Nowrouzi A, et al. Adeno-associated virus vector genomes persist as episomal chromatin in primate muscle. Journal of virology 2008;82:7875-85.
2. Barde I, Laurenti E, Verp S, et al. Regulation of episomal gene expression by KRAB/KAP1-mediated histone modifications. Journal of virology 2009;83:5574-80.
3. Ferreira RC, Freitag DF, Cutler AJ, et al. Functional IL6R 358Ala allele impairs classical IL-6 receptor signaling and influences risk of diverse inflammatory diseases. PLoS Genet 2013;9:e1003444.
4. McFarland-Mancini MM, Funk HM, Paluch AM, et al. Differences in wound healing in mice with deficiency of IL-6 versus IL-6 receptor. Journal of immunology (Baltimore, Md : 1950) 2010;184:7219-28.
5. Xu B, Chen Q, Yue C, et al. Prognostic value of IL-6R mRNA in lung adenocarcinoma and squamous cell carcinoma. Oncol Lett 2018;16:2935-48.
[References]

OC 31.1

Whole Exome Sequencing of Patients Treated with Adeno-associated Virus Serotype 8-Factor IX (AAV8-FIX) Gene Therapy Reveals Potential Determinants of Persistent Transgene Expression

I. Bilic1, P. Monahan2, V. Berg3, F. Scheiflinger1, B.M. Reipert1
1Baxalta Innovations GmbH, a Takeda Company, Vienna, Austria, 2Spark Therapeutics, Philadelphia, United States, 3IMC University of Applied Sciences Krems, Krems, Austria

Main Topic: Hemophilia and Bleeding (including Transfusion)
Category: Hemophilia Gene Therapy

Background: The clinical phase I/II study investigated BAX335, an AAV8-based gene therapy for FIX replacement in patients with hemophilia B. BAX335 was well-tolerated in all patients with no clinical thrombosis and no evidence of inhibitors or other FIX-directed immunity. Persistent therapeutic FIX activity of 20% without bleeding or replacement therapy was achieved in only one patient for >4 years (Index patient). In all other patients, FIX activity declined after 5-11 weeks and could not be rescued by corticosteroid treatment.
Aims: Identification of genetic predispositions as molecular determinants for long-term transgene expression in the Index patient using whole exome sequencing (WES).
Methods: High-coverage WES of genomic DNA from patients treated with BAX335 was combined with state-of-the-art variant identification and prioritization. Index patient was compared to the remaining seven patients to identify variants potentially mediating persistent transgene expression. Prioritized variants were evaluated according to the predicted deleteriousness using CADD, PolyPhen-2 and SIFT algorithms and to the phenotypic manifestations of their target genes.
Results: Two of the identified variants found in the index patient could impact transgene expression. The first are compound heterozygous variants of the nuclear receptor co-repressor complex SMRT/NCOR2, which mediates transcriptional silencing by tethering general chromatin-modifying enzymes to target genes. Since chromatinized episomal AAV-transgene is regulated by histone modifications1,2, the identified SMRT variants highlight protection from heterochromatinization as one of the mechanisms for ensuring persistent transgene expression.
The most indicative variant was a heterozygous missense mutation in the interleukin-6 receptor gene (IL-6R). IL-6R haploinsufficiency is well documented3-5, thus the identified variant might decrease sensitivity to IL-6-mediated inflammatory stress caused by BAX335 (AAV8-capsid and/or transgene) and protect targeted hepatocytes.
Conclusions: Two important parameters required for successful liver-targeting gene therapy are implicated: (i) design of robust expression cassette resistant to gene silencing by heterochromatinization and (ii) protection of targeted hepatocytes from inflammatory stress induced by viral load.


Ref. Nr. Reference
1. Penaud-Budloo M, Le Guiner C, Nowrouzi A, et al. Adeno-associated virus vector genomes persist as episomal chromatin in primate muscle. Journal of virology 2008;82:7875-85.
2. Barde I, Laurenti E, Verp S, et al. Regulation of episomal gene expression by KRAB/KAP1-mediated histone modifications. Journal of virology 2009;83:5574-80.
3. Ferreira RC, Freitag DF, Cutler AJ, et al. Functional IL6R 358Ala allele impairs classical IL-6 receptor signaling and influences risk of diverse inflammatory diseases. PLoS Genet 2013;9:e1003444.
4. McFarland-Mancini MM, Funk HM, Paluch AM, et al. Differences in wound healing in mice with deficiency of IL-6 versus IL-6 receptor. Journal of immunology (Baltimore, Md : 1950) 2010;184:7219-28.
5. Xu B, Chen Q, Yue C, et al. Prognostic value of IL-6R mRNA in lung adenocarcinoma and squamous cell carcinoma. Oncol Lett 2018;16:2935-48.
[References]

Code of conduct/disclaimer available in General Terms & Conditions

By clicking “Accept Terms & all Cookies” or by continuing to browse, you agree to the storing of third-party cookies on your device to enhance your user experience and agree to the user terms and conditions of this learning management system (LMS).

Cookie Settings
Accept Terms & all Cookies