Generation Bio Co. announced data from a study achieving tolerability and targeted factor VIII expression levels in hemophilia A mice with a single dose of closed-ended DNA (ceDNA) delivered via the company’s novel, cell-targeted lipid nanoparticle (ctLNP) system. In this study, conducted with a ceDNA development construct, a dose response was observed across three cohorts, with the highest dose of 2.0 mg/kg yielding a mean human factor VIII expression of 23% of normal. The company also announced data from studies conducted with a ceDNA research construct delivered via ctLNP demonstrating approximately 2:1 species translation from mice to non-human primates (NHPs). All doses in mice and NHPs were well-tolerated up to the highest dose of 2.0 mg/kg. These data confirm delivery of ceDNA to the liver via ctLNPs in higher species. Generation Bio has previously demonstrated in immunocompetent mice that its ceDNA constructs with human factor IX achieved durable expression for months and that expression increased proportionately with redosing. In parallel with hemophilia A, Generation Bio plans to advance programs in phenylketonuria (PKU) as well as in additional rare and prevalent diseases that are addressable using the company’s liver-specific ctLNP delivery system and established, capsid-free manufacturing. Translation from mouse to NHP was established by delivering the same weight-adjusted dose of ceDNA-ctLNP in each species. Two separate studies were conducted. In the first study, a mean human factor VIII expression level of ~1% of normal was observed in mice at day 7 following a single dose of a ceDNA research construct delivered systemically via a liver-directed ctLNP at 1 mg/kg. This translated to a mean human factor VIII expression level of ~1% of normal in NHPs at day 5 using identical material and weight-adjusted dosing (1 mg/kg). In a second study employing a similar ctLNP, a mean human factor VIII expression level of ~3% of normal was observed in mice at day 5 following a single dose of a ceDNA research construct at 2 mg/kg. This translated to a mean factor VIII expression level of 1% of normal in NHPs at day 5 using identical material and weight-adjusted dosing at 2 mg/kg. In totality, the 2:1 expression ratio from mice to NHPs is similar to that observed across other modalities delivered via LNPs, including RNAi and mRNA, and establishes a basis for final development candidate selection. The expression levels in mice using a ceDNA development construct, coupled with the 2:1 expression ratio from mice to NHPs and expected 1:1 translation from NHPs to humans, support selection of a final hemophilia A clinical development candidate this year. The observed expression levels in mice with a ceDNA development construct may correspond to or exceed the 5% of normal threshold in humans that has been clinically proven to prevent serious bleeds in patients. Doses of ceDNA-ctLNP in both mice and NHPs were well tolerated up to 2 mg/kg, the highest dose evaluated. There were no adverse clinical observations, changes in clinical pathology, or histopathology findings including in the liver and spleen in NHPs. Generation Bio’s ctLNP employs N-acetyl galactosamine, or GalNAc, as the ligand for targeting of liver cells via the asialoglycoprotein receptor, or ASGPr. GalNAc-ASGPr is a well-validated, selective ligand-receptor pair for systemic delivery to hepatocytes. In previously released data from a study in immunocompetent mice, a single intravenous dose of ceDNA formulated in an LNP yielded long-term expression in the liver for months using the reporter protein luciferase and human factor IX. LNP delivery does not stimulate an antibody response, thereby enabling redosing and overcoming a major limitation of viral gene therapy. Generation Bio has previously released data showing that ceDNA delivered in an LNP does not induce neutralizing antibodies and can be redosed in mice with normal immune systems. Redosing five weeks after the initial dose proportionately increased expression using both the reporter protein luciferase and human factor IX in mice. After the first administration of a factor IX ceDNA research construct in an LNP, mice demonstrated 5% to 10% activity levels of factor IX protein in the blood. After repeat administration at the same dose, the activity levels rose to 10% to 20%. These results support the potential of Generation Bio’s non-viral gene therapy platform to safely titrate patients to the desired level of protein expression and to enable repeat dosing if needed to maintain expression over a lifetime.