801.

GENE THERAPY AND TRANSFER | NOVEMBER 13, 2019

The Factor VIII Variant X5 Enhances Hemophilia a

Gene Therapy Efficiency By Its Improved Secretion
*,1 *,1 *,2 *,2
Franziska Horling, PhD, Johannes Lengler, PhD, Wenjing Cao, PhD, Biao Dong, PhD,
*,3 *,1 3
Bagirath Gangadharan, Maurus De La Rosa, PhD, Werner Hoellriegl, PhD, Birgit
*,1 *,3 *,2,4 *,5
M Reipert, PhD, Friedrich Scheiflinger, Weidong Xiao, PhD, Hanspeter Rottensteiner
1
Drug Discovery Austria, Baxalta Innovations GmbH, a Takeda company, Vienna, Austria
2
Sol Sherry Thrombosis Research Center, Temple University Medical School, Philadelphia, PA
3
Baxalta Innovations GmbH, a Takeda company, Vienna, Austria
4
Ivygen, Philadelphia, PA
5
Baxalta Innovations Gmbh, A Member of the Takeda Group of Companies, Vienna, Austria

bloodjournal Blood blood (2019) 134 (Supplement_1) : 3356.

http://doi.org/10.1182/blood-2019-126489

Introduction. Adeno-associated virus (AAV)-based factor VIII (FVIII) gene therapy holds great promise
to provide clinical benefit in patients with hemophilia A. However, very high doses are currently needed
to achieve therapeutic factor levels and the durability appears to be limited to a couple of years. Vector
efficiency could be improved by employing more potent liver-specific promoters, but this might come at
the price of overstraining the cellular protein folding capacity, causing FVIII to misfold in the lumen of
the Endoplasmic Reticulum (ER). This event would in turn activate the unfolded protein response, cause
oxidative stress, and if not resolved may even induce cell death.

Aims. The objective of the presented study was to test whether the B-domain deleted (BDD)-FVIII-X5
variant can overcome the secretion challenge of high level FVIII expression in the context of hepatic gene
therapy.

Methods. The human FVIII variant BDD-FVIII-X5 harboring 5 amino acid exchanges in the A1 domain
was previously isolated in a screen aimed at identifying those residues in porcine FVIII that are critical for
efficient secretion. BDD-FVIII and BDD-FVIII-X5 were produced in Chinese Hamster Ovary (CHO) cells
and purified to apparent homogeneity using standard procedures. The preparations were assayed for
total protein by UV absorbance at 280 nm and FVIII activity by a chromogenic assay. Both FVIII variants
were vectorized using AAV8 and tested in the human liver cell line HepG2 and FVIII knockout mice
(E17) at various doses. Resulting samples were assayed for FVIII chromogenic activity. The potential
immunogenic risk was evaluated in three hemophilic mouse strains (E17, human FVIII transgenic,
humanized HLA-DRB1*1501).

Results. A characterization of purified recombinant Refacto-like BDD-FVIII and the corresponding X5

variant revealed similarity of the two proteins and their specific activities in particular, indicating that
introduction of the 5 amino acids from porcine FVIII did not alter functionality of human BDD-FVIII. In
vitro expression of BDD-FVIII-X5 in a human liver cell line resulted in substantially increased FVIII activity
levels in the supernatant compared with the non-modified BDD-FVIII, commensurate with enhanced
secretion of the X5 variant. Intravenous delivery of liver-targeted AAV8 vectors carrying the BDD-FVIII-X5
transgene achieved substantial increases in plasma coagulation activity over BDD-FVIII in FVIII-deficient
mice, even when highly efficient codon-optimized F8 nucleotide sequences were employed. Evaluation of
the immunogenicity of the BDD-FVIII-X5 variant by an immunological risk assessment did not reveal any
increased immunogenic risk compared to BDD-FVIII.

Conclusions: The fully active BDD-FVIII-X5 variant demonstrated improved secretion in vitro and in
vivo, resulting in substantially higher FVIII levels in a hemophilia A mouse model. No signs of enhanced
immunogenicity were noted in a comparative immunogenicity study. The results obtained warrant further
exploration of the BDD-FVIII-X5 variant for a next generation hemophilia A gene therapy.

Disclosures
Horling: Baxalta Innovations GmbH, a Takeda company: Employment. Lengler: Baxalta Innovations
GmbH, a Takeda company: Employment. Gangadharan: Baxalta Innovations GmbH, a Takeda
company: Employment. De La Rosa: Baxalta Innovations GmbH, a Takeda company: Employment,
Equity Ownership. Hoellriegl: Baxalta Innovations GmbH, a Takeda company: Employment, Equity
Ownership. Reipert: Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership.
Scheiflinger: Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Xiao:
Ivygen: Other: Patent application on FVIII-X5 has been submitted. Rottensteiner: Baxalta Innovations
GmbH, a Takeda company: Employment, Equity Ownership.

Author notes
*Asterisk with author names denotes non-ASH members.

© 2019 by the American Society of Hematology