Identification and Characterization of STAC-BBB, an Engineered AAV Capsid That Exhibits Widespread Transduction of the Central Nervous System in Cynomolgus Macaques

Matthew Tiffany, Stephanye Frias, Lori Andrews,Ankitha Nanjaraj, Russell Darst,

Stephen Wist, Satria Sajuthi,Yuri Bendaña, Hung Tran, Sarah Mueller, Bryan

Zeitler, Amy M. Pooler, David S. Ojala

ASGCT 27th Annual Meeting - May 8, 2024

Disclosure

I am a full-time employee of Sangamo Therapeutics

2

SIFTER platform leverages cell type specific measurement of capsid-mediated transgene expression

AAV library administered

to cyno macaques

Capsids reach brain tissue

Traditional methods

stop here

Overcome biological barriers

Transduce neurons

Neurons

Astrocytes

Pericytes

BMECs

Express transgene in neurons

Delivery goal

polyA

Thousands of UMIs per barcode

UMI

At least 4 barcodes per capsid

Barcode

Transgene

Promoter

hSyn1 p40

or

Capsid

modifications

Capsid Library A

Readouts

DNA ✓

UMI 1

…

UMI N

Barcode 1

Barcode 2

Barcode 3

Barcode 4

CMV

Capsid Library B

Capsid Library C

SIFTER = Selecting In vivo For Transduction and Expression of RNA

mRNA ✓ Cell type ✓

3

Multiple library screening rounds were conducted to identify STAC-BBB

Round 1

Round 2

Round 3

Individual evaluation

60,000 capsids with

1,260 capsids with at

Single best capsid

least 4 unique sequence

100 million

sequence replicates.

variant (STAC-BBB) with

replicates. Includes

capsids screened.

Includes controls with

reporter gene or

controls with known

known performance.

therapeutically-relevant

performance.

ZF cargo.

4

Study design for Round 3 SIFTER library selection

Objective: Determine relative performance of 1,260 capsid variants and select lead variant for individual evaluation

Capsid

Cargo

Multiplexed promoters

1,260 Capsid Variant Library

hSyn1 promoter

CMV promoter

(Neurons only)

(All cells)

Dose & Duration

Species

5E13 vg/kg

Cynomolgus

Macaque

N=3 animals

3-week study

5

STAC-BBB is the top performing capsid in the round 3 library for neuronal transduction

644-fold enrichment in brain

Log2 Fold Change

in Neuronal mRNA

Expression (Y-axis):

Enrichment score relative to the administered library

Larger value is better

Whole Brain

Assessment

hSyn1 neuron specific promoter

High

performing

capsids

Coefficient of Variation (X-axis):

Variation in performance across tissue

samples that were evaluated

Smaller value is better

Neuronal RNA expression (3-week study, hSyn1)

Data averaged from all three animals

STAC = Sangamo Therapeutics AAV Capsid

Unique Molecular Identifier count (Color):

Informs number of unique AAV transduction events

Darker green is better

Fraction of replicates found (Bubble size): Informs consistency of replicate recovery

Larger circle is better

6

STAC-BBB is the top performing capsid in the round 3 library for neuronal transduction

644-fold enrichment in brain

Log2 Fold Change

in Neuronal mRNA

Expression (Y-axis):

Enrichment score relative to the administered library

Larger value is better

Whole Brain

Assessment

hSyn1 neuron specific promoter

Coefficient of Variation (X-axis):

Variation in performance across tissue

samples that were evaluated

Smaller value is better

Neuronal RNA expression (3-week study, hSyn1)

Data averaged from all three animals

Unique Molecular Identifier count (Color):

Informs number of unique AAV transduction events

Darker green is better

Fraction of replicates found (Bubble size): Informs consistency of replicate recovery

Larger circle is better

7

STAC-BBB exhibits 700-fold higher neuronal mRNA expression relative to AAV9

Assessment across brain slices and animals

STAC-BBB

Published capsid

AAV9

hSyn1 neuron specific promoter

Brain Slice No.

2 4 6 8 10 12 2 4

6 8 10 12 2 4

6 8 10 12

Animal 1

Animal 2

Animal 3

Neuronal RNA expression (3-week study, hSyn1)

Box represents 25th - 75th percentile of library performance. Whiskers are 1.5x the interquartile range.

8

STAC-BBB mediates higher neuronal mRNA expression in all CNS regions

Cortex

Hippocampal

Deep

Brain

Spinal

inChangeFold

ExpressionmRNA

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Brain

Cerebellum Stem

Cord

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  • = AAV9 not found; * = STAC-BBB not found

Neuronal RNA expression (3-week study, hSyn1)

All three animals on study

150 brain punches analyzed

STAC-BBB Published capsid AAV9

hSyn1 neuron specific promoter

9

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Hippocampal

Deep

Brain

Peripheral

Cortex

Region

Brain

Cerebellum Stem DRG

Tissues

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150 brain punches analyzed

STAC-BBB

Published capsid

AAV9

CMV ubiquitous promoter (All cells)

STAC-BBB is de-targeted

from DRGs and

* = AAV9 not found

peripheral tissues

Ubiquitous RNA expression (3-week study, CMV)

All three animals on study

10

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Sangamo Therapeutics Inc. published this content on 08 May 2024 and is solely responsible for the information contained therein. Distributed by Public, unedited and unaltered, on 08 May 2024 22:46:06 UTC.