Sangamo Therapeutics : Widespread CNS Tau Knockdown for the Potential Treatment of Alzheimer’s Disease and other Tauopathies

Introduction

Highly potent and specific ZFRs targeting MAPT

MAPT exon 1

ZFR1 reduces MAPT expression at the single-neuron level over a 100-fold dose range in vivo

STAC-BBB enables whole CNS tau knockdown in NHPs

Figure 2. Initial screen for ZFRs targeting human MAPT

384 ZFRs were designed to target human MAPT and screened by transient transfection in SK-N- MC cells. ~29% of ZFRs achieved at least 50% repression of the MAPT transcript.

Vehicle

3E8 VG

3E9 VG

VG

MAPT in ZFR+

Neun+ cells

spots per area ZFR+ NeuN+ cells

Adult Cynomolgus

Macaques

+

STAC-BBB

hSYN1

ZFR1

28 d

ISH,

IHC

1E14 VG/kg 2E13 VG/kg 5E12 VG/kg Vehicle

Pons

MAPT normalized to the mean of ATP5b, EIF4A2 and scaled to the mean of control-treated animals

A ZFR genomic medicine for the potential treatment of AD and other tauopathies

Candidate

ZFR1

3E10

Multiplexed RNAscope ISH / IHC assay for NeuN, MAPT mRNA, and ZFR mRNA.

N=5-6 htau mice treated with Vehicle or AAV9.hSYN1.ZFR1 at 3E8, 3E9, or 3E10 VG/hemisphere.

% MAPT in

Average 11,379 cells analyzed per mouse

Figure 5. Single-cell analysis of ZFR+ neurons in the hippocampus of htau mice

A dose response for neuronal transduction and hippocampal coverage was observed across a 100-fold AAV dose range as assessed by multiplexed ISH/IHC (see Fig. 4 for study details). At all

ZFR

MAPT gene

MAPT mRNA

STAC-BBB

tau

Figure 3. Characterization of a potent, specific ZFR candidate targeting human MAPT

ZFR1 reduced MAPT in human iPSC-derived neurons by >95% with no off-targets. In mouse primary cortical neurons, ZFR1 was highly specific, but showed no repression of mouse Mapt due to a single mismatch in the mouse target site.

ZFR1 stably lowers tau mRNA and protein by >90%

doses tested, individual neurons expressing ZFR1 showed >95% human MAPT reduction. ZFR1 therefore achieves potent single-neuronMAPT knockdown over a wide range of expression levels.

Widespread knockdown of human MAPT following IV delivery of ZFR1 to aged htau mice

STAC-BBB

Cervical spinal cord

Temporal cortex

Vehicle

STAC-BBB

in vivo after hippocampal delivery to htau mice

ZFR mRNA

VehicleZFR

Vehicle

Thalamus

Figure 1. AAV ZFR for tauopathies

A ZFR targeting the tau gene is packaged into STAC-BBB, which crosses the BBB with a one-time IV administration.The ZFR stably represses MAPT transcription in neurons, resulting in specific reduction

3 mo old

htau1+mice

AAV9

hSYN1

ZFR1

/ ng RNA

ns

Expression

MAPT mRNA

Normalized levels

Total tau protein

10 mo old htau mice

+

AAV.PHP.B

hSYN1

ZFR1

8 mo

NeuN MAPT NeuN p-tau

STAC-BBB

Vehicle

STAC-BBB

of tau protein. Lowering neuronal tau allows for clearance of all aberrant tau protein forms, improved neuron function and survival, and reduced susceptibility to

1. mo

1. mo

Transcripts

% Normalized

%

RNA ISH, IHC

ZFR MAPT

Multiplexed RNAscope ISH / IHC assay for: NeuN, MAPT mRNA, and ZFR mRNA (pons); NeuN, ChAT, MAPT mRNA, and ZFR mRNA (cervical spinal cord);

NeuN, S100β, MAPT mRNA, and ZFR mRNA (precentral gyrus, temporal cortex, thalamus). Images from a representative NHP dosed at 1e14 vg/kg, 28 days post administration:

Figure 7. Bulk and single-cell ZFR expression and MAPT repression across the NHP CNS

ZFR1 was delivered intravenously to adult NHPs using a novel BBB-penetrant capsid, STAC-BBB. Bulk

tau mediated toxicity.

RNA, ISH, protein IHC

MAPT normalized to the mean of Atp5b, Eif4a2, Gapdh and scaled to

the mean of the vehicle group for each region.

Representative images from the hippocampus of 18 mo old htau mice treated with Vehicle or ZFR High dose. Multiplexed RNAscope ISH / IHC assay for: NeuN, MAPT mRNA, and ZFR mRNA; an adjacent section was stained for p-tau/NeuN.

RT-qPCR analysis of 220 punches per NHP showed dose-dependent ZFR expression throughout the entire brain at all dose levels. Dose-dependentMAPT repression was observed in many regions

Conclusions and next steps

(Fig. 4) (Fig. 5)

htau1 mice harbor the full-length human MAPT locus on a Mapt -/- null background. N=5-7 mice per group. MAPT normalized to mean of Atp5b, Eif4a2, Gapdh and scaled to the vehicle treated group for each time point. Total tau normalized to total protein concentration and scaled to the vehicle treated group for each time point. Mixed-effects analysis: Within treatment comparisons3 vs 6 mo for each group (upper); Within timepoint comparisons to baseline (arrow) (lower). ns, not significant; * P<0.05, **** P<0.0001.

Figure 6. Reduction of human MAPT throughout the brain of aged htau mice

including the pons and thalamus. MAPT expression from non-neuronal cells contributes to this bulk signal. Single-cell analysis revealed widespread ZFR expression and neuron-specificMAPT repression

• We characterized a candidate ZFR that represses human MAPT by >95% with no off targets.
• Potent ZFR-mediatedMAPT repression was demonstrated at the bulk and single-cell level in the mouse and NHP CNS. ZFR treatment reduced total tau and p-tau in htau mice.
• ZFR treatment was well tolerated in all models, doses, and time points tested.
• This work supports the initiation of an IND-enabling GLP toxicology study for a STAC-BBB delivered ZFR for the potential treatment of tauopathies, including AD and PSP.

Select images made with Biorender.com

Figure 4. Reduction of human MAPT in the hippocampus of htau transgenic mice

htau1 mice received dual, bilateral intrahippocampal injections of AAV9 hSYN1-ZFR1 at 3E8, 3E9, or 3E10 VG per hemisphere. Cohorts of mice were euthanized at 3 mo or 6 mo post injection. A clear dose response for ZFR expression, MAPT repression, and total tau protein knockdown was observed, ranging from ~50 to >90% at both the 3 mo and 6 mo timepoints. The treatment was well tolerated and there were no test‐-article related histopathology findings.

1 Andorfer C. et al. J Neurochem. 2003;86(3):582 590.

10 mo old htau mice received tail vein injections of AAV.PHP.B hSYN1-ZFR1 at 1.2E13, 3.5E13, or 1.2E14 VG/kg. Mice were euthanized 8 mo after injection. A dose response for ZFR expression and MAPT repression was observed in all 9 brain regions analyzed, with ~50-70% reduction at the highest dose in most regions. Single-cell analysis using multiplexed ISH/IHC revealed potent MAPT repression across the brain, including reduction of hyperphosphorylated p-tau in hippocampal neurons.The treatment was well tolerated and there were no test-article related histopathology findings.

throughout the brain and all spinal cord levels.This includes regions critical for AD and PSP.

Acknowledgments and disclosures

We would like to thank our previous collaborators, including Biogen and the laboratory of Dr. Brad Hyman.We also thank all current and former Sangamo colleagues that have contributed to the tau program.This work was funded by Sangamo Therapeutics.