Development of a neuroinflammatory systems biology platform to enable discovery of disease-modifying therapeutics for Alzheimer’s disease

Lauren H. Martens1, Kelley C. Larson1, Jonathan M. Levenson1, Sarah Kolitz2, Matthew Ung2, Rebecca Kusko2, Jermaine Ross2, Michael Marconi1, Rob Nunes1, Christopher D. Dejesus1, Ben Zeskind2, Tom Miller1, Barbara Tate1

1Tiaki Therapeutics, 700 Main Street, Cambridge, MA 02139 USA;2Immuneering, 245 Main Street, Cambridge, MA 02142 USA

The presence of neuroinflammation is a pathological hallmark of multiple CNS degenerative diseases. GWAS and transcriptomic studies identified microglial dysfunction as a key driver of the chronic neuroinflammation associated with Alzheimer’s disease. Characterization of the non-innate immune cell functions of microglia during CNS development revealed the requirement of neuron-glia interactions for normal brain function. These insights provide potential therapeutic strategies whereby adult microglial function is modulated to protect the aging brain. In vitro assays fail to model the complex CNS biology present in vivo, including authentic neuron-glia interactions and aspects of human aging. Tiaki Therapeutics has developed a systems biology platform that models the neuroinflammatory aspect of Alzheimer’s disease and other neurodegenerative disorders. An integral part of Tiaki’s system is an adult ex vivo brain slice assay, which permits the longitudinal analysis of all CNS cell types within their authentic matrix and inter-cellular environment. Transcriptomes were obtained for whole tissue and isolated microglia to establish the neuroinflammatory signature of the platform. Following an initial period of dynamic changes in gene expression, the Tiaki platform exhibits a stable neuroinflammatory state that can be monitored by transcriptomic and protein analyses. Using a bioinformatics approach, the gene expression signature observed in the adult ex vivo brain slice assay significantly correlates to gene expression data from Alzheimer’s patients. Tiaki’s assay is uniquely positioned to provide longitudinal transcriptomic and proteomic signatures for CNS health and disease, as well as biomarkers for target engagement and compound efficacy. To date we have screened chemical matter for a number of targets in our platform and have been able to identify targets that mitigate the inflammatory signature. Our model of the chronic neuroinflammation associated with Alzheimer’s disease enables the discovery and development of disease-modifying therapeutics against microglial targets that demonstrate benefit to surviving neurons.

ABSTRACT

BACKGROUND

Tiaki Systems Biology Platform Reveals Novel Targets for Neuroinflammatory AD

Tiaki systems biology platform contains all relevant cell types in their native environment in a state that models neuroinflammatory AD

Healthy and inflamed microglia characterized(gene expression, protein, secretome)

Longitudinal characterization of microglial influence on neuronal

health

Adapted from: Neurology September 17, 2013; 81 (12) CLINICAL IMPLICATIONS OF NEUROSCIENCE RESEARCH, Microglia: Multiple roles in surveillance, circuit shaping, and response to injury, Eduardo E. Benarroch

Adapted from Lue et al. Journal of Neuropathology and Experimental Neurology 1996Schneider et al. Journal Alzheimers Disease 2009

SantaCruz et al. Journal of Neuropathology and Experimental Neurology 2011Boyle et al. Frontiers Aging Neuroscience 2013

Equal Aβ plaque deposition in AD

and PWD

Patients with AD/dementia exhibit

significant numbers of microglia with

activated morphology surrounding Aβ

plaques

Tiaki targets eliminate inflammation and restore cognitive function

Neuroinflammation: A Critical Driver of Cognitive Decline in AD

• Adult microglia, neurons and other glia, with functional synaptic networks

• Transcriptome, proteome, secreted biomarkers, functional endpoints

AD Healthy

Human data sets:• Validated with human

genetics• Healthy & Diseased• Genotype & Clinical

phenotypes• Pathology/ATN profiles

Human Data

TiakiData

Novel targetsPatient population selectedBiomarker strategy defined

BioinformaticsAnalysis

Tiaki ex vivo platform Curated human data sets

Tiaki Approach to Translational ScienceMETHODS

RESULTSTiaki Ex Vivo Platform Models Human AD Refe

rence

A

Targ

et A

Targ

et B

-1.5

Cluster 1

Cluster 2

Cluster 3-1.0

-0.5

0

0.5

CONCLUSIONS

FUNDING

• Tiaki has created a systems biology platform that models the transcriptomic signature observed in patients with Alzheimer’s disease.

• The disease signature is reversible, indicating the platform is suitable for use in target identification and validation efforts.

• The disease signature can be reversed by pharmacological agents that interfere with the normal function of distinct targets that lie in different functional pathways.

• Target A is in active drug discovery, with initial clinical tests anticipated in 2021.

Ex Vivo System Neuroinflammatory Signature is Reversed by Interfering with a Non-obvious Target

• Differential expression (DE) analysis of RNAseq data from postmortem, cognitively normal controls and pathology confirmed AD human brain tissue (data transformed)

• Significant correlation between DE of AD patients and Tiaki system comparing orthologous differentially expressed genes

• Pleiotropic reference treatment tested in Tiakiplatform demonstrates the system can be modulated to reverse the human AD disease signature

Transformed fold change values

Ref A

TiakiAD

VEHICLE

TGFb

2_25

0 ng/m

L

PF543

_400

nM

CYM5035

8_50

0 nM

0

2000

4000

6000

8000

MMP12

Sec

rete

d M

MP

12

(pg/

ml)

* p<0.02

Log2 (Fold C

hange)

Ref A

Targ

et A

Targ

et B

• Dysregulation of a signaling pathway reveals potential targets for interventiono Targets A and B are both drug-able nodes in a

pathway dysregulated in both AD and the Tiakisystem

o Modulation of Target A reverses the transcriptomic AD disease signature similar to Reference A, while Target B does not fully reverse the signature

• MMP12 secretion is reduced by treatments that reverse the AD transcriptomic signature

• Suggests MMP12 as PD biomarker for compound efficacy

Ref A

Targ

et B

Targ

et A

Vehic

le

MMP12

* *

Correlation Target A vs AD: rho -0.18, p < 0.0001

• Dementia Discovery Fund: Seed stage funding

Bulk transf

TGF

12039587796

115134153172191210229248267286305 -1.0

-0.5

0

0.5

1.0

Tiaki

ADTiaki

124477093116139162185208231254277300323346369392 -1.0

-0.5

0

0.5

1.0

Ex Vivo System Signature Not Responsive to NSAID Treatment

Targ

et 2

Vioxx

-0.4

Log2 (Fold C

hange)

Cluster 1

Cluster 2

Cluster 3

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

VioxxReference A

• Comprehensive transcriptomic analysis indicates Reference A treatment suppresses inflammatory gene expression (top cluster), increases expression of genes involved in neuronal and synaptic function (middle cluster), and suppresses neurodegenerative genes (lower cluster)

• Treatment of the Tiaki system with the COX-2 inhibitor, Vioxx, does not reverse the human AD disease signature

Vehicle

Viox

x0

50

100

150

200

% o

f M

atc

hed

Veh

icle

[Mean± S

EM

]

MMP12 • Matrix metalloproteases are secreted by activated microglia and result in detrimental downstream effects for neurons

• MMP12 expression is increased in AD and the Tiaki system

• Vioxx treatment does not alter the secreted biomarker MMP12

Treatment of slices with Vioxx significantly reduces PGE2 release into the media, confirming target

engagement. (data not shown)

Correlation Reference A vs AD: rho −0.29, p < 0.0001

Ex vivo adult brain culture

Proprietary transcriptomic signature of neuroinflammation

Protein assays for neuronal

health

Secreted protein panels to monitor inflammatory signaling

Tiaki Organotypic Slice Assay Provides Datasets Spanning Multiple Cell Types and Endpoints

Ref A

Ref A