PB0146 - Functional Characterization of a Human F9 Gene-inserted Product in Hemophilia B Mice

Sunday, June 25, 2023

18:30 – 19:30 ET

Room: Exhibition

Presenting Author(s)

Dan Chalothorn, PhD

Director in Cardiovascular and Renal Research
Regeneron Pharmaceuticals, Inc.
Tarrytown, New York, United States

Co-Author(s)

LS

Leah Sabin

Executive Director, Regeneron Genetics Medicines
Regeneron Pharmaceuticals, Inc.
Tarrytown, New York, United States
KL

KehDih Lai

Manager Cardiovascular Research
Regeneron Pharmaceuticals, Inc.
Tarrytown, New York, United States
YK

Yong Kim

Senior R&D specialist
Regeneron Pharmaceuticals, Inc.
Tarrytown, New York, United States
RS

Rachel Sattler

Lead R&D Specialist
Regeneron Pharmaceuticals, Inc.
Tarrytown, New York, United States
CM

Catherine Moroski-Erkul

Scientist
Intellia Therapeutics, Inc.
Cambridge, Massachusetts, United States
KW

Kathryn Walsh

Senior Scientist
Intellia Therapeutics, Inc.
Cambridge, Massachusetts, United States
CS

Cindy Shaw

Director
Intellia Therapeutics, Inc.
Cambridge, Massachusetts, United States
JP

Jonathan Phillips

Vice President
Intellia Therapeutics, Inc.
Cambridge, Massachusetts, United States
DG

David Gutstein

Vice President, Strategic Program Direction – Hematology
Regeneron Pharmaceuticals, Inc.
Tarrytown, New York, United States
CK

Christos Kyratsous

Senior Vice President, Research
Regeneron Pharmaceuticals, Inc.
Tarrytown, New York, United States
LM

Lori Morton

Senior Vice President, Research
Regeneron Pharmaceuticals, Inc.
Tarrytown, New York, United States

Background: Hemophilia B is an X-linked genetic disorder resulting in reduced activity of coagulation Factor IX (FIX). Regeneron and Intellia are jointly developing a novel liver-targeted therapeutic modality for hemophilia B using CRISPR/Cas9 to insert human F9 into the Albumin locus to restore FIX activity.

Aims: To characterize functional activity of mouse-generated human FIX (hFIX) with functional assays and to evaluate the ability of mouse-generated hFIX to restore hemostasis in mice with hemophilia B.

Methods: Humanized Albumin mice with F9 deficiency (Alb hu/hu ; F9 -/-) were dosed with rAAV8‑F9 and a lipid nanoparticle (LNP) containing both mRNA for Cas9 and single guide (sg) RNA targeting the first intron of human ALB. Six weeks post-treatment, plasma was collected from these mice and evaluated for function using clinically validated one-stage clotting and two-stage chromogenic substrate FIX activity assays. In addition, Alb hu/hu ; F9 -/- mice were subjected to a tail bleeding challenge to assess the function of mouse-generated hFIX in restoring hemostatic capacity.

Results: Mouse-generated hFIX had similar specific activities as purified human FIX (from human plasma) in the respective ex vivo one-stage clotting and two-stage chromogenic substrate FIX activity assays, and the activities were concentration dependent. In addition, hFIX produced in Alb hu/hu ; F9 -/- mice was functionally active and able to rescue hemostasis relative to non-treated Alb hu/hu ; F9 -/- mice following a tail bleeding challenge.

Conclusion(s): Our targeted gene insertion platform uses CRISPR/Cas9 to insert human F9 into the Albumin locus and yields a hFIX product that is functionally indistinguishable from purified human FIX in ex vivo FIX activity assays and is capable of rescuing a bleeding phenotype in Alb hu/hu ; F9 -/- mice. This therapeutic strategy represents a novel potential treatment for patients with hemophilia B.