Investigating Critical Challenges in the Gene Therapy Field Mapping out the Landscape & Discussing Future Strategies Sander van Deventer Managing Partner Forbion Capital Partners CSO and General Manager uniqure Amsterdam Prof. Translational Gastroenterology Leiden University Medical Center
Make a safe and effective product That can treat all patients That can be manufactured at scale at at a reasonable cost of goods
Improving the efficacy of gene therapy in humans Capsid stoichiometry Promotors hyperactive proteins Translating efficacy and safety from animal studies (mouse) to the clinical setting End-organ vector distribution and expression Liver Brain Gene Therapy for Gene Silencing Immunology Humoral and cellular responses Pre-existing antibodies Re-treatment Manufacturing
Data!
Vector design determines therapeutic efficacy Vector-host interactions Capsid (Higher vector delivered to appropriate cell types) Promoter (Higher gene expression in appropriate cell types) Gene (Higher gene expression and/or higher protein specific activity)
Improving the efficacy of gene therapy in humans Capsid stoichiometry Promotors hyperactive proteins Translating efficacy and safety from animal studies (mouse) to the clinical setting End-organ vector distribution and expression Liver Brain Gene Therapy for Gene Silencing Immunology Humoral and cellular responses Pre-existing antibodies Re-treatment Manufacturing 6
High potency capsids produced in insect cells - optimal VP1:2:3 stoichiometry - WO 2007/046703 Improved AAV vectors produced in insect cells WO2015/137802 Further improved AAV vectors produced in insect cells cap genes need to be engineered to achieve correct stoichiometry and required potency of the vector
Capsid modifications lead to a ± log increase in potency (FIX in primates) Conclusion: AAV capsids of the same serotype can have widely different efficiencies 8
AAV Capsid optimization via directed evolution Selection x 6 Input AAV Capsid Sequences Validation of selected variants Diversity Generation of Variants Package into Functional Vectors
Improvement of vectors: the power of directed evolution Representative example of technology licensed by uniqure from 4D therapeutics (USA): significant improvements of vectors within reach
Reporter: SEAP Vector: AAV Promoters: Synthetic, liver selective Selectivity: Validated Activity: Dynamic range, up to 45 times greater than control
Hyperactive mutant proteins AMT-060 LP1 promoter SV40 intron hfixco PolyA 1 ~ 2.4 kb -46 PP Light Chain AP Heavy Chain 415 R338L AMT-061 AGG toctg LP1 promoter SV40 intron hfixo-padua PolyA -46 ~ 2.4 kb 1 R338L PP Light Chain AP Heavy Chain 415 A single amino acid change from arginine to leucine at position 338 (R338L)
% h F I X p r o t e i n ( % o f n o r m a l h u m a n ) FIX-Padua shows ~6-fold higher FIX activity in non-human primates h F I X p r o t e i n a v e r a g e p o s t d o s e ( d a y 4 - w k 2 5 ) 2 0 1 5 1 0 5 0 A M T - 0 6 0 5 e 1 2 A M T - 0 6 1 5 e 1 2
Improving the efficacy of gene therapy in humans Capsid stoichiometry Promotors hyperactive proteins Translating efficacy and safety from animal studies (mouse) to the clinical setting End-organ vector distribution and expression Liver Brain Gene Therapy for Gene Silencing Immunology Humoral and cellular responses Pre-existing antibodies Re-treatment Manufacturing 14
Mouse liver tissue Non-human Primate liver tissue
PBS AAV5-hFIXco 5 E 12 gc/kg AAV5-hFIX Padua 5 E 12 gc/kg AAV5-hFIX Padua 2.5 E 13 gc/kg AAV5-hFIX Padua 9 E 13 gc/kg % cells positive for hfix Assessment of transduction efficacy in liver of Non Human Primates: AAV5-PaduaFIX 50 40 30 20 10 0
Improving the efficacy of gene therapy in humans Capsid stoichiometry Promotors hyperactive proteins Translating efficacy and safety from animal studies (mouse) to the clinical setting End-organ vector distribution and expression Liver Brain Gene Therapy for Gene Silencing Immunology Humoral and cellular responses Pre-existing antibodies Re-treatment Manufacturing 20
s e c o n d s L a t e n c y t o f a l l ( s ) s e c o n d s R6/2 HD mice R 6 / 2 H D m i c e ( 1 2 w e e k s o f a g e ) 7 5 * 3 0 T i m e t o t o u c h h i n d l e g s 5 0 2 0 2 5 1 0 0 P B S + 5 % S u c r o s e L o w M i d H i g h A A V - m i H T T 0 4 0 T i m e t o c l a s p 3 0 2 0 1 0 0 W T v e h i c l e R 6 / 2 v e h i c l e R 6 / 2 L o w R 6 / 2 M i d R 6 / 2 H i g h A A V - m i H T T Lisa Spronck, Astrid Valles-Sanchez, Taneli Heikkinen, Martin de Haan, Harald Petry, Pavlina Konstantinova and Melvin Evers ESGCT 2017
Improving the efficacy of gene therapy in humans Capsid stoichiometry Promotors hyperactive proteins Translating efficacy and safety from animal studies (mouse) to the clinical setting End-organ vector distribution and expression Liver Brain Gene Therapy for Gene Silencing Immunology Humoral and cellular responses Pre-existing antibodies Re-treatment Manufacturing 22
Pre-existing neutralizing antibodies to AAV-capsids Interfere with end-organ transduction Preclude re-administration of gene therapy (children) (recall) T cell responses to AAV-capsid proteins expressed by transduced cells May cause organ damage Reduce transgene expression Immune responses to (CpG) elements in the transgene DNA (?) Humoral immune responses to expressed proteins
Prevalence of Serum IgG and Neutralizing Factors Against Adeno-Associated Virus (AAV) Types 1, 2, 5, 6, 8, and 9 in the Healthy Population: Implications for Gene Therapy Using AAV Vectors Boutin et al 2010 24
Luciferase assay Red text, FIX activity 1.3% 6.8% Positive by GFP assay No correlation between neutralizing activity detected in vitro by luciferase and clinical outcome Negative 3.0% AMT-060-01 patients Transduction with AMT-060 was successful in patients with titers as high as the 90th percentile of those seen in the healthy population
Experimental set-up proof of concept in NHPs
SEAP and hfix mrna expression Mean Reduction levels NABs by immuno-adsorption: 11-fold SEAP and hfix protein expression
Improving the efficacy of gene therapy in humans Capsid stoichiometry Promotors hyperactive proteins Translating efficacy and safety from animal studies (mouse) to the clinical setting End-organ vector distribution and expression Liver Brain Gene Therapy for Gene Silencing Immunology Humoral and cellular responses Pre-existing antibodies Re-treatment Manufacturing 29
Preferred technology for: Protein replacement Silencing (brain, eye, heart) Gene editing Paradigm shifts needed: Regulatory Reimbursement Upfront payments Annuity payments Pay for performance Clinical medicine Centralized treatment centers Rare diseases Not so Rare diseases Common diseases