Inducible systems for gene therapy medicines Graham Whyteside, - - PowerPoint PPT Presentation

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Inducible systems for gene therapy medicines Graham Whyteside, - - PowerPoint PPT Presentation

Jan 11, 2024 322 likes •589 views

Improving human health by enabling safer, more effective cell and gene medicines through proprietary genomics, bioinformatics and intelligent data driven design. Inducible systems for gene therapy medicines Graham Whyteside, Principal

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Inducible systems for gene therapy medicines

Graham Whyteside, Principal Scientist

Improving human health by enabling safer, more effective cell and gene medicines through proprietary genomics, bioinformatics and intelligent data driven design.

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Constitutive Expression

Tissue-selective Variable strength Multi-tissue control Size to specification

Regulated Gene Control

Pharmacologically responsive Oral Control

Inducible Repressible Tissue selective Safety switch

Intelligent Design for Greater Gene Control

Pharmacologically responsive Oral Control

Dually regulated Multi-gene control Pathway control

Autoregulatory, Environmental Control

Biologically responsive Adaptive Control

Self-regulated Temporally regulated Physiologically regulated

Enabling the development of new gene medicines

Center of Excellence for Gene Regulation

Promoters for current products Promoters for future gene medicines

i ii iii iv

Company Development

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Tissue specific inducible promoters

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Cell and Gene Medicine

POC of design model: Liver specific induction

  • Liver specific
  • Small molecule inducible
  • GRAS/FDA
  • Modularised enhancer
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Cell and Gene Medicine

POC of design model: Liver specific induction

50 100 150 Muscle Spleen Heart Brain Kidney Liver

Tissue expression level of inducible TF

Protein mRNA

Liver specific Small molecule inducible Modularised Enhancer

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Cell and Gene Medicine

POC of design model: Liver specific induction

  • Identified 4 different enhancers

responsive to small molecule

  • Tested inducibility of each enhancer
  • Huh7 model
  • Different levels of induction for each

enhancer

  • Dose responsive
  • Further tested in primary hepatocytes

01 02 03 04 5×106 1×107 1.5×107

SYNP-LIND- Normalised RLU 50nM 150nM

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SLIDE 7

Cell and Gene Medicine

POC of design model: Liver specific induction

01 02 03 04 50 100 150 200

RLU normalised SYNP-LIND-

Primary Hepatocytes

Enhancer 1 was chosen as our lead candidate

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Cell and Gene Medicine

POC of design model: Liver specific induction

  • Created combinations of enhancer 1 with

different minimal promoters

  • Inducibility
  • Strength
  • Background
  • Level of induction and strength dependent
  • n MP
  • >5-fold range from single enhancer

01 08 12 16 50 100 150

SYNP-LIND- RLU normalised Uninduced Induced

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Cell and Gene Medicine

POC of design model: Liver specific induction

  • Enhancer 1
  • Self-contained 51-bp module
  • Test modularity

01 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 CMV-IE 500 1000 1500 2000

SYNP-LIND- RLU normalised Uninduced Induced

MP1 MP2 MP3 MP4

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Cell and Gene Medicine

POC of design model: Liver specific induction

  • Lead candidates
  • Robustness of promoter
  • Expression of EPO

SYN-LIND-01 SYN-LIND-06 CMV_IE 20 40 60 80 100

[EPO]mIU/mL

SYN-LIND-01 10 20 30 40

[EPO]mIU/mL Conc 2 Conc 3 No Drug Conc 1

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Cell and Gene Medicine

POC of design model: Liver specific induction

  • Are the promoters specific?
  • Tested in HEK293
  • Promoters show no activity in HEK293

cells

  • Indicates initial analysis of specificity was

correct

01-Primary 01-HEK293 06-Primary 06-HEK293 2 4 6

Ratio to CMV-IE Uninduced Induced

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Cell and Gene Medicine

POC of design model: Liver specific induction

  • Lead candidates
  • Cloned into AAV vector
  • Effect of ITR’s
  • Inducibility and background

SYNP-LIND-01 pAAV-LIND-01 pAAV-01 Primary SYNP-LIND-06 pAAV-LIND-06 pAAV-06-Primary

1 2 3 4 5 6

Ratio to CMV-IE Drug No Drug

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Cell and Gene Medicine

POC of design model: Liver specific induction

  • Can the promoters be switched off?
  • Removal of drug after 24hrs
  • Drug removal returns activity to

baseline

  • Progressed to in vivo POC

pAAV-LIND-01 pAAV-LIND-06 CMV-IE

100 200 300 400 500

SYN-LIND Ratio to CMV-IE Induced Uninduced Drug removed

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Cell and Gene Medicine

Days after injection Fold change

35 40 45 20 40 60

SYN-LIND-01 SYN-LIND-06

POC of design model: Liver specific induction

30 35 40 45 109 1010 1011

Days after injection

Luciferase expression (photons/second)

SYNP-LIND-01 SYNP-LIND-06

Specific Inducible 12-24hr induction >45-fold induction

SYNP-LIND-01 SYNP-LIND-06 2×109 4×109 6×109

Luciferase expression (photons/second) 9 24 48

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Cell and Gene Medicine

POC of design model: Liver specific induction

Part 3 SYNP-LIND-01

  • Enhancer optimisation
  • 3 component parts
  • Optimised each part
  • Improved activity
  • Enhanced in vivo performance?

Part 2 Part 1

SYNP-LIND-01 SYNP-LIND-20 SYNP-LIND-21 SYNP-LIND-22 SYNP-LIND-23 SYNP-LIND-24 50 100 150

Novel variants of LIND promoter

Normalised RLU No drug Inducer

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  • Synpromics have developed
  • Pipeline for identifying and constructing tissue specific inducible promoters
  • Novel liver-specific inducible promoter
  • Single input system
  • GRAS/FDA approved drug
  • Small enhancer
  • 51-bp
  • Increases options for packaging in AAV
  • 4x CMV-IE possible from a 200bp promoter
  • Modular and configurable
  • Can be multiplied to increase activity
  • Different strengths/background
  • Modify the enhancer
  • Change MP
  • Dynamic range of 16-fold at fully induced activity

Overview

Cell and Gene Medicine

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Tissue specific repressible promoters

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Cell and Gene Medicine

POC: Liver repressible promoters

  • Liver specific
  • Small molecule inducible
  • GRAS/FDA
  • Modularised repressor
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Cell and Gene Medicine

POC: Liver repressible promoters

  • Identified a liver specific repressible TF
  • Modular component
  • 29bp
  • Engineered Synpromics Liver specific

promoters to contain module

  • Modular component confers

repressibility on the promoter

  • 1 module is adequate for repression

Base promoter

01 02 03 04 Control 2 4 6

Ratio to CMV-IE

Primary Unt Primary Treat

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Cell and Gene Medicine

POC: Liver repressible promoters

  • Optimised expression
  • Analysis of liver transcriptomics
  • Identified 10 5’ UTR designs
  • All <50 bp long
  • Improved activity of base promoter

SYNP-OFF-03

  • 1 promoter design up to 15 fold CMV-

IE

  • Promoters will be tested in vivo summer

2019

01 02 04 10 03 11 13 06 08 09 12 05 07 5 10 15 20

SYNP-OFF Normalised to CMV-IE No Drug Drug UTR designs

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  • Synpromics have developed
  • Pipeline for identifying and constructing tissue specific repressible promoters
  • Novel liver-specific repressible system
  • Single input system
  • GRA/FDA approved drug
  • Small module
  • 29-bp
  • Increases options for packaging in AAV
  • Can be added to existing promoters to add repressibility
  • Discovered Liver 5’ UTRs
  • Small <50bp
  • Can increase dynamic range of promoter (3-fold)
  • Do not interfere with repression

Overview

Cell and Gene Medicine

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Victoria Torrance Anne Braae Simon Waddington Rajvinder Karda Riccardo Privolizzi

Synpromics Controlled gene expression in the liver: design of constitutive, inducible and repressible promoters for use in gene medicine Metabolic, Storage, Endocrine, Liver and Gastrointestinal Diseases II | 798 | Wednesday 1st May, 5 – 6pm UCL Development of Novel Promoters for Neurological Gene Therapy Neurologic Diseases II | 558 | Tuesday 30th April, 5 – 6pm Solid Biosciences Identification of Novel Muscle-Specific Promoters for AAV Gene Expression in Skeletal and Cardiac Muscles Musculo-skeletal Diseases | 822 | Wednesday 1st May, 5 – 6pm uniQure Biopharma B.V. Towards AAV5-Mediated Gene Therapy for Hemophilia A with a Factor IX Variant that Functions Independently of FVIII AAV Vectors and Disease Targets II | 959 | Thursday 2nd May, 11:45am – 12pm uniQure Biopharma B.V. Development of an AAV5-Based Gene Therapy for Fabry Disease AAV Vectors and Disease Targets II | 960 | Thursday 2nd May, 12pm – 12:15pm

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If you would like to know more about Synpromics and gene control technology, please get in touch! info@synpromics.com www.synpromics.com Roslin Innovation Centre Easter Bush Campus Midlothian, EH25 9RG

Co Contact Us

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Cell and Gene Medicine Introduction Company Development PromPT Cell and Gene Medicine Bioprocessing Applications Partnering

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