Sangamo Therapeutics : Single-cell characterization of ST-506, a BBB-penetrant epigenetic repressor of Prion protein expression, in the nonhuman primate brain

Published: 2026-05-12 06:20:08 ET SGMO

Published on 05/12/2026 at 06:20 am EDT

Cell segmentation

Neuron identification

ZFR mRNA detection

detection

Single-cell characterization of ST-506, a BBB-penetrant epigenetic repressor of prion protein

expression, in the nonhuman primate brain

Shih-Wei Chou, Daniel Chung, Satria Sajuthi, Andrew Olin, Garrett Lew, Annemarie Ledeboer, Madelena Nguyen, Kenneth Kennard, Marina Falaleeva, Sarah Mueller, Andrew Young,Yanmei Lu, Gregory Davis, Gill Atkinson, Kathleen Meyer, David Ojala, Bryan Zeitler

Sangamo Therapeutics, Inc., Richmond, CA

Poster #1469

Neuron-targeted ZFR and

STACTM-BBB capsid

One-time IV

administration

Zinc Finger

Repressor (ZFR)

STACTM-BBB capsid

PrPC

PRNP

mRNA

PRNP gene

Stable PrP reduction in neurons in the brain

ST-506

1E +14 vg/kg

28 days in-life

ISH + IHC

50 µm

20 µm

Vehicle ST-506

50 µm

20 µm

Vehicle ST-506

Superior Frontal

ST-506

Vehicle

ST-506

NeuN+ cell

NeuN- cell

ST-506

5 µm

nucleus

cytosol

B PRNP expression in single neurons

Fix hemispheres, mount sections and permeabilize

Hybridize target-specific RNAscope probes

Amplify signal through sequential hybridization of amplifier and label probes

40 Gyrus

DAPI NeuN ZFR

% NeuN+ with ZFR+

NeuN ZFR

Region selection. DAPI-based cell detection and 5-µm expansion.

Neurons were classified using a random forest model trained using cell shape features and NeuN (purple) expression as

ZFR transcripts (green) within cell objects were detected as spots using a subcellular tool. Circles (orange) highlight individual spots identified in each cell. ZFR+ and ZFR- cells were

PRNP transcripts (white) within cell objects were detected as spots using a subcellular tool. Circles (magenta) highlight

12500

Median PRNP

spot total intensity

10000

7500

5000

2500

****

↓ ****

95.9%

Vehicle ST-506 ST-506

Prion disease is a rapidly progressive and fatal neurodegenerative disorder driven by the

conformational conversion of the normal cellular prion protein, PrPC, into its misfolded, pathogenic

predictors

Vehicle

ST-506

classified using a random forest model trained using cell and spot features.

individual spots identified in each cell.

ZFR+

ZFR-

5 Detect

fluorescent labels

isoform, PrPSc. There are over 1,500 new cases each year in the US, Europe, and Japan, and no currently available disease-modifying treatments. Genetic, biochemical, and pathological evidence supports PrPC reduction as a primary therapeutic strategy. We are advancing our clinical candidate, ST-506, for the treatment of prion disease. ST-506 is a one-time intravenously delivered, AAV based neuron-specific gene therapy that employs a zinc finger repressor (ZFR) directed towards the human prion gene (PRNP) packaged in the blood-brain barrier (BBB) penetrant capsid STACTM-BBB, to achieve sustained brain-wide downregulation of PrPC. Previously, we demonstrated that post-symptomatic treatment of RML-inoculated mice with a neuron-targeted surrogate ZFR profoundly extended survival (>494 days post-inoculation) and reduced PrP for >17 months (Figure 1). Here, we evaluate the effect of ST-506 in nonhuman primates (NHP) in suppressing PrP expression at the

Combine IHC for cell type ID

Quantification via QuPath

(A) Schematic diagram of NHP study design. (B) Schematic diagram showing the experimental workflow from sample preparation, in situ hybridization (ISH), IHC, imaging, and quantification. (C-E) Transduction of ST-506 was assessed by RNAscope (ZFR, PRNP) multiplexed with IHC (NeuN). Representative confocal images from superior frontal gyrus (C) and hippocampus (D) reveal ZFR expression (green) in NeuN+ neurons (purple) in an ST-506-treated animal but not vehicle-treated animal. (E) A custom-trained classifier was used to quantify ZFR+ cells in the superior frontal gyrus. In the ST-506 treatment, 33.9% of neurons were classified as ZFR+, compared to 0.29% in vehicle treatment. The total

(A) Step-wise single-cell quantification using Qupath. (B) Median PRNP spot total intensity in NeuN+ cells classified as ZFR+ or ZFR-. N = 7422, 1736, 3392 NeuN+ cells for vehicle, ST-506 ZFR+, and ST-506 ZFR-, respectively. **** P < 0.0001 with Mann Whitney test. Plotted values correspond to fluorescent intensity measurements from cells in superior frontal gyrus. Median ± 95% CI.

single-cell level.

1 mm

A B

PHP.B

NeuN+ cells analyzed were 5128 and 7466 for ST-506 and vehicle treatments, respectively.

RML

hSYN1-mZFR

119 dpi

100

Vehicle

MST 169.5 dpi

ST506_ZFR+ (n=1736)

ST506_ZFR- (n=3392)

Vehicle_ZFR- (n=7422)

Median normaliezed PRNP

spot total intensity per cell

5×10 -7

Uninoculated High dose

% Survival

MST >494 dpi

Mid dose

NeuN ZFR

DAPI ZFR

MST 462 dpi

A Ventral hemisphere B Superior frontal gyrus C

NeuN PRNP

DAPI PRNP

PRNP ZFR

4×10 -7

25 µm

DAPI NeuN ZFR

DAPI NeuN PRNP

3×10 -7

2×10 -7

1×10 -7

NeuN PRNP

DAPI PRNP

0 25 50 75 100 125 150 175 200

Median normalized ZFR

Vehicle AAV-ZFR

Low dose

ST-506

MST 340 dpi

0 100 200 300 400 500

Days post inoculation (dpi)

spot total intensity per cell

(A) Representative ISH/IHC images show heterogeneous ZFR expression with uniformly low PRNP levels in individual neurons. Arrows in blue, red, and yellow are cells with high, low, and no ZFR detection, respectively. (B) PRNP mRNA expression in the

Immunohistochemistry (IHC) analysis showed reduced anti-PrP staining (black) in the brains of mice treated with PHP.B-hSYN1-mZFR (1E+14 vg/kg) after 518 days compared to vehicle treatment in wildtype mice. (B) Median survival time (MST) of RML-inoculated mice was extended from 169.5 dpi in vehicle-treated animals to 340, 462, and >494 dpi for low (1E+13 vg/kg), mid (3E+13 vg/kg), and high (1E+14 vg/kg) dose treated animals, respectively.

Human iPSC-derived neurons

Human PRNP mRNA

125

100

MOI

ST-506 ZFR

% Normalized

PRNP expression

3.3E2

1.0E3

3.3E3

1.0E4

3.3E4

1.0E5

3.3E5

The ST-506 ZFR induced PRNP mRNA repression across a wide dose range in human iGABA neurons.

AAV6 21 days

STACTM-BBB is a trademark of Sangamo Therapeutics, Inc. Disclosures:This work was fully funded by Sangamo Therapeutics, Inc.

PRNP ZFR

5 mm

50 µm

25 µm

50 µm

25 µm

200 µm

25 µm

Vehicle

NeuN PRNP

NeuN ZFR

NeuN PRNP

NeuN ZFR

NeuN PRNP

DAPI PRNP

DAPI ZFR

DAPI PRNP

Representative fluorescent images from ST-506- (top panel) and vehicle- (bottom panel) treated NHP brain sections stained for ZFR transcripts (green), PRNP transcripts (white), NeuN neuronal marker (purple), and DAPI (blue) demonstrate widespread ST-506-mediated PRNP reduction. (A) Ventral coronal section and enlarged views of (B) superior frontal gyrus, (C) hippocampus, and (D) thalamus1 show neurons expressing ZFR (green dots) lack detectable PRNP (white dots) in an ST-506-treated animal, but not in the vehicle control. 1NeuN staining in the thalamus was below detection and requires further optimization.

Presented at ASGCT 2026

superior frontal gyrus is shown as the median normalized PRNP spot total intensity per cell (y-axis) after correcting for imaging batch effects and cell size. Values were binned into 20 equal quantiles. In ZFR- neurons, PRNP expression is comparable between vehicle- (black line) and ST-506-treated (blue line) samples, indicating minimal PRNP repression in the absence of detectable ZFR. In contrast, ZFR+ neurons from ST-506-treated samples (green line) exhibit robust PRNP repression at nearly all measured ZFR expression levels. Notably, over 55% of ST-506-treated ZFR+ neurons display near-zero PRNP expression, regardless of increasing ZFR spot total intensity per cell, demonstrating potent PRNP suppression across neuronal ZFR expression levels.

ZFRs mediated potent PrP reduction and significantly extended survival in RML-inoculated mice when dosed after symptom onset.

Previously, we showed that ST-506 mediates bulk mRNA and protein knockdown throughout the brain in NHPs at levels comparable to those associated with substantial survival extension in RML mice.

Using our in-house ISH/IHC assay to enable single-cell assessment of ST-506 biodistribution and prion repression in the NHP brain, we demonstrated PRNP expression was reduced by >95% in ST-506 expressing neurons.

Widespread ST-506 expression was observed throughout the NHP brain.

ST-506 dosed at 1E+14 vg/kg was well tolerated in NHP over 28 days of follow up.

The potent and specific PRNP repression in NHP combined with the good tolerability support the potential of ST-506 for the treatment of symptomatic prion disease in the clinic.

Acknowledgements: RNAscope support from Advanced Cell Diagnostics, Inc. Imaging support from Molecular Device, Inc, Keyence Corporation of America, and Zeiss. Some illustrations were created using BioRender.com.

Disclaimer

Sangamo Therapeutics Inc. published this content on May 12, 2026, and is solely responsible for the information contained herein. Distributed via Public Technologies (PUBT), unedited and unaltered, on May 12, 2026 at 10:19 UTC.