A first-in-class splice sensor assay

Lucerna, Inc. is a biotech company with a proprietary fluorescent aptamer technology developed by a team of experts in RNA biology. We are located in the Downstate Technology Incubator, Brooklyn, NY.

The Spinach™ Technology has highly versatile applications:• Drug Discovery – modular platform, mix-and-read, HTS assays• RNA Imaging – Live-cell imaging of endogenous mRNAs and non-coding RNAs• R&D Toolkits – Customized RNA tools for molecular detection and diagnostics

Spinach™ is the RNA version of the green fluorescent protein (GFP). It is genetically encodable with proven utility for imaging RNA in living cells and measuring cellular metabolites in HTS assay format.

Who

Why

What

Introducing Lucerna, Inc.

Pre-Clinical

• H3 Biomedicine (cancer)

• PTC Therapeutics (Familial dysautonomia)

• PTC Therapeutics (Huntington’s disease)

Clinical Trials

• Roche / PTC (Spinal muscular atrophy)

Approved

• Biogen / Ionis (Spinal muscular atrophy)

• Sarepta Therapeutics (Duchenne muscular atrophy)

Splice Modulating Therapeutics

Compare Current Methods to Spinach™ Assay

RT-PCR/qPCR PNA-based FRET RNA-seq Mini-gene splice reporter

Spinach™ splice assay

High-throughput

Fast

Simple

Requires optimization

Instrumentation Simple Complex Complex Simple Simple

Mix-and-read

Endogenous mRNA

Translation-independent

Pre-mRNA

Exon 1 Exon 2 Exon 3

Intron 1 Intron 2

Exon 1 Exon 3Skips exon 2

Exon 1 Exon 2Skips exon 3

Splice sensor:

Sensor dye:

Target recognition region

Green fluorescenceNo fluorescence

Correct splicingExon 1 Exon 10Exon 1 Exon 2

Incorrect splicingExon 3

Lucerna™ Splice Sensor Assay

Lucerna has a phase I grant to develop a HTS assay for new cancer therapeutics What is pyruvate kinase (PKM)?

It is the enzyme that catalyzes the final step in glycolysis

Forms two isoforms: PKM1 (exon 10 skipping) and PKM2 (exon 9 skipping)

What happens in the cell?

In normal healthy cells, PKM1 is expressed

In cancer cells, PKM2 is expressed to produce the needed energy

Why PKM2 isoform is a good target?

PKM2 knockdown - reduces tumor formation and increases apoptosis

PKM2 knockdown - enhances anti-cancer drug efficacy

Targeting PKM2 proteins alone cannot completely block tumorigenic activities

Splice Sensor Assay for Cancer Therapeutics

PKM1 splice sensor fluorescence: 30-fold selective increase in the presence of

PKM1 RNA (normal isoform) PKM1 splice sensor response is fast and

stable

PKM2 splice sensor fluorescence 300-fold selective increase in the presence

of PKM2 RNA (cancerous isoform) PKM2 splice sensor response is fast and

stable

Lucerna™ Splice Sensor Fluorescence

PKM2 Splice Sensor

Demonstrates high selectivity and sensitivity toward PKM2 RNA

Active over a broad range of PKM2 RNA concentrations

PKM2 Splice Sensor is Selective and Sensitive

9“Spinach” “Corn” “Radish”“Broccoli”“Eggplant”

Spinach™ Aptamers in Other Colors

10

LucernaTM Splice Sensor Platform Summary

Fast, Selective, Sensitive

Stable assays, over 12 hours

Customizable, Target any RNA Isoform of Interest

Study designs for endogenous mRNAs, no artificial systems

Versatile, HTS Assays, Mix-and-read

Applications for biochemical and cell-based assays

Multiplexing with multiple colors and wavelengths

Adapted from Scotti et al., Nature Reviews Genetics 17,19–32(2016)

Diseases Caused by Aberrant Splicing

Disease Gene(s) affected Splicing effectMuscular:

Dilated cardiomyopathy LMNA, RBM20 Gene target mis-splicing, exon extension

Duchenne muscular dystrophy DMD Frameshift

Spinal muscular atrophy SMN1 Altered protein stability

Neuro:

ALS TARDP, FUS Gene target mis-splicing

Leukodystrophy ADLD Gene target mis-splicing

Parkinson disease PINK1, MAPT, ATP6AP2 Increased exon inclusion, exon skipping

Other:

Cancer BCL-x, BRAF, CD44, PKM, VEGF….. Various mechanisms

Lipodystrophy LMNA Intron retention

Progeria LMNA Exon truncation

Opportunities with the Splice Sensor Technology

Partner Drug Discovery Platform – partner the technology platform to co-develop a drug discovery assay for internal R&D

Partner Biotech Toolkits – by licensing the technology for a wide range of biotech toolkits in the near term for development and commercialization.

Partner Research Platform - by licensing the splice sensor platform as a research tool to institutions, pharma and biotech companies.

Website:Lucerna, Inc.TM

Contact:Karen Wue: karen.wu@lucernatechnologies.comt: 646.504.5697

Location:Downstate Technology Center760 Parkside Avenue, Suite 327B

Brooklyn, NY 11226

Lucerna, Inc.TM - Thank You

www.lucernatechnologies.com