Designed Zinc Finger Protein Transcription Factors for Single-Gene Regulation Throughout the Central Nervous System

Transcription

1 Designed Zinc Finger Protein Transcription Factors for Single-Gene Regulation Throughout the Central Nervous System Bryan J Zeitler 1 *, Sarah L DeVos 2 *, Susanne K Wegmann 2 *, Kimberly Marlen 1, Qi Yu 1, Hoang-Oanh Nguyen 1, Annemarie Ledeboer 1, David S. Ojala 1, Lei Zhang 1, David A. Shivak 1, Jeffrey C Miller 1, Edward J Rebar 1, Brigit E Riley 1, Bradley T Hyman 2, Michael C Holmes 1 1 Sangamo Therapeutics 2 Mass General * These authors contributed equally to this work.

2 Disclosure I am an employee of Sangamo Therapeutics

3 Single-administration AAV ZFP to lower all tau forms at the DNA level Zinc Finger Protein Transcription Factor (ZFP-TF) Non-integrating AAV Vector Stable repression of Tau/MAPT gene Packaging into AAV Administration options: Intracranial, Intrathecal or Intravenous ZFP-TFs reduce tau at the DNA level 3

4 Outline CNS Disease 1. ZFP-TF technology 2. Targeting tau for the treatment of tauopathies 3. Repression of mouse and human tau in vitro Gene Regulation

5 Outline CNS Disease 1. ZFP-TF technology 2. Targeting tau for the treatment of tauopathies 3. Repression of mouse and human tau in vitro Gene Regulation

6 Zinc Fingers can be engineered to recognize specific DNA sequences Most abundant DNA binding domain in the human genome amino acid peptide Binds 3-4 bp of DNA Specificity and affinity can be engineered 6

7 ZFP-TFs can be engineered to regulate any gene 7

8 ZFP-TFs can be engineered to regulate any gene Fully human-derived sequence 3 ZFPs can fit in 1 raav genome High potency 2 targets per cell 8

9 Outline CNS Disease 1. ZFP-TF technology 2. Targeting tau for the treatment of tauopathies 3. Repression of mouse and human tau in vitro Gene Regulation

10 Tau tracks closely with clinical symptoms and neuronal loss Cortical loss tau PET Amyloid PET Amyloid b plaques Extracellular tau tangles Intracellular Tau defects causes many other tauopathy diseases: Frontal Temporal Dementia Progressive Supranuclear Palsy Corticobasal Degeneration Chronic Traumatic Encephalopathy Xia C Dickerson BC. Association of In Vivo [18F]AV-1451 Tau PET Imaging Results With Cortical Atrophy and Symptoms in Typical and Atypical Alzheimer Disease. JAMA Neurol. February 20,

11 Neuronal loss in tauopathies is blocked by tau reduction Pathogenic tau Phospho-tau Oligomer NFT Multiple tau species Pathogenic form(s) unclear Disease Prevent Reverse Lowering tau Healthy neuron Axonal tau Diseased neuron Pathological, mislocalized tau Aggregates and tangles Dying neuron Consumed by tau pathology Atrophied 11

12 Single-administration AAV-ZFP-TF to lower all tau forms at DNA level Zinc Finger Protein Transcription Factor (ZFP-TF) Non-integrating + ZFP-TF - ZFP-TF AAV Vector Packaging into AAV Routes of Administration: Intracranial, Intrathecal or Intravenous ZFP-TFs repress tau at the DNA level Reduced tau prevents neurodegeneration 12

13 Outline CNS Disease 1. ZFP-TF technology 2. Targeting tau for the treatment of tauopathies 3. Repression of mouse and human tau in vitro Gene Regulation

14 High ZFP density design for rapid lead identification 185 ZFP-TFs = Neuro2A cells were harvested after 24 hours and analyzed by RT-qPCR. 14

15 Tau mrna High ZFP density design for rapid lead identification 185 ZFP-TFs = 0 tau mrna 100 ZFP-TF mrna / txn 1000 / 300 / 100 / 30 / 10 / 3 ng ZFP A ZFP B ZFP C ~30% of ZFP-TFs from the initial screen reduced tau by 50% Neuro2A cells were harvested after 24 hours and analyzed by RT-qPCR. 15

16 Highly specific ZFPs can be identified at the initial design stage tau ZFP A tau ZFP B 4 0 tau ZFP C 0 0 Down > 2x Up >2x Neuro2A ng ZFP mrna txn / 24 hrs / n=4-6 biological replicates 16

17 Altering Zinc Finger-Phosphate Contacts to Improve Specificity DNA Zinc Finger Most zinc fingers make a conserved DNA phosphate contact from their β-sheet Conserved Arg PO 4 contact Alteration of this contact may improve ZFP specificity Elrod-Erickson et al., Structure AAY.pdb

18 P Value (-log10) P Value (-log10) Phosphate contacts modulate global specificity tau mrna tau Neurons 15 + ZFP-D microarray 10 5 AAV9 ZFP-D tau d qrt-pcr microarray ZFP-D R Q microarray E5 / 1E5 / 3E4 / 1E4 AAV9 VG per cell Microarray dose: 1E5 VG per cell, n=4-6 biological replicates; primary mouse cortical neurons 18

19 P Value (-log10) Potent, single-gene specific repression of tau in primary neurons Neurons + Mouse tau mrna Mouse tau protein tau Global specificity N=26,491 transcripts evaluated AAV9 ZFP-E 7d 1 % 2 % Mock ZFP E Mock ZFP E qrt-pcr ELISA microarray Fold change Microarray dose: 1E5 VG per cell, n = 4-6 biological replicates primary mouse cortical neurons; Mouse Clariom D Genechip 19

20 % Normalized Expression P Value (-log10) Single-gene specific tau repression in human ips neurons ips Neurons + Target site alignment ZFP-E Human Tau mrna tau Global specificity N=19,959 transcripts evaluated Hu ACCCACCAGCTCCGGCAC **** 47% AAV6 ZFP-E Mo ACCCACCAGCTCCAGCAC 19d qrt-pcr microarray Fold change **** P < , n = 5-7 biological replicates. Dose: 1E5 VG per cell. Human Clariom S Genechip. 20

21 ZFP-TFs are a potent and specific tau-targeted therapay Activity Potent tau reduction in mouse and human neurons Tau repression persists for at least 11 months in the hippocampus ZFP-TFs can reduce tau by up to 80% across the brain Specificity ZFPs with no off-targets can be efficiently engineered Single-gene specific tau reduction in the hippocampus following IV delivery Efficacy ZFP-treatment reduced APP/PS1 neuritic dystrophies by 50% across the cortex 21

22 Sangamo Research Mass General Kim Marlen Qi Yu Oanh Nguyen Annemarie Ledeboer David Ojala Mohammad Samie Rainier Amora Tanja Meyer Kathy Meyer Brigit Riley Michael Holmes Lei Zhang David Shivak Jeff Miller Sarah Hinkley Stephen Lam Yuri Bendana Dave Paschon Ed Rebar Alicia Goodwin Hung Tran Tim Gabriele Andrea Kang Richard Surosky Sarah DeVos Susanne Wegmann Danny Mackenzie Caitlin Commins Brad Hyman