TUNR - Gene Expression Control System | NanoString Expression Analysis | Custom Gene Engineering Service...

1. Product/Service Overview

2. Corporate Headquarters
• Established in 2016 in St. Louis
• STL is a major hub for start-ups and early
stage tech
• An extremely cost-effective region for
business
• Utilization of the BioGenerator Labs –
part of the Cortex District
• State-of-the-art specialized equipment
• Access to core facilities at Washington
University in St. Louis

3. EDWARD WEINSTEIN, PHD
Founder, President & CEO,
and Director
• PhD, Genetics, Harvard University
• 16 years of industry experience in pharma and research tools
• Co-Founder and President of startup in research tools space
successfully exited in 2014
• Previously: President, Horizon Discovery; President, SAGE Labs;
Director, Sigma Aldrich
CRYSTAL WINKELER, PHD
Founder & COO
DAVID SMOLLER, PHD
Founder & Chairman
of the Board
• PhD, Molecular Cell Biology, Washington University
• Experience in due diligence, company creation and investments
• Previously: Sr. Analyst, BioGenerator
• PhD, Biology, Emory University
• Founded and successfully exited 3 startups in research tools
space
• Broad experience in startup and public company leadership
• Current: General Partner, Cultivation Capital | Previously: CEO,
SAGE Labs; CSO, Sigma Aldrich
Canopy Management | Background Information
A team built upon leadership with success in the research tools space

4. In-Licensing Technology
• Canopy focuses on finding in-licensing
opportunities for new technologies rather
than in-house discovery
• Goal of building a pipeline of technologies
to license
• Create a balanced portfolio of products
and services in various states of maturity
• Universities, research institutions,
pharmaceutical and biotechnology
companies

5. Focus: Whole Genome Editing/Whole Genome
Monitoring
• Provide cutting edge tools and services for modifications to genes – any edit,
any genome
• Build technologies for whole genome monitoring and support them with the
highest level of service

6. TUNR Biology
Precisely control protein expression levels
• Insertion of polyA tracks results in
decreased protein expression
• Translation machinery stalls on polyA
track
• Ribosome stalling regulates translation
• Stalling occurs because positively
charged lysines from polyA stretch
stalls/interacts with negatively charged
ribosome exit channel
• The strength of the stall is dependent on
the number of lysines
• Stalled ribosome is recognized by
mRNA surveillance pathways
• Stalled mRNA ribosome is cleaved and
incomplete protein product is degraded
Inser&on of polyA tracks results in decreased protein expression
Adapted from: Arthur, L. L. et al. 2017
Nature Communica<ons

7. Advantages
Adapted from: Arthur, L. L. et al. 2017
Nature Communica<ons
• TUNR allows for wild type, knockout,
and everywhere in between
• TUNR does not effect protein function
• Predictable, robust, precise
• Evolutionarily conserved mechanism
WT
(AAA)6
(AAA)9
(AAA)12
Human Cancer Cells
WT (AAA)6 (AAA)9 (AAA)12

8. Cell Lines Expressing mCherry
CHO (Chinese Hamster Ovary)
HEK 293(Human Embryonic Kidney)
In this case mCherry is dox-induced
PolyA track control is independent of promotor strength.
Translational control by lysine-encoding A-rich sequences

9. Functional Assays
• Chloramphenicol acetyltransferase (CAT) gene confers resistance to the antibiotic
chloramphenicol (CAM) in a dose dependent manner
• Regulated CAT protein by inserting polyA tracks.
• Longer polyA tracks reduce ability to grown in high CAM conditions
E. Coli CAT Functional Assay

10. à Fine tune control of
endogenous gene expression
à Stability
à Reproducibility and precision
à Artifact elimination
TUNR
Cas9
sgRNA
st
TUNR + CRISPR

11. Technology Summary
Applications
• Examine downstream effects of target
TUNing
• Study genes critical for survival in vitro
• Study embryonic lethal knockouts
• Rescue experiments
11
Examine downstream effects of target TUNing
Investigate embryonic lethal genes
TUNR
Downstream Effects
DT2
Target
DT1 DT3

12. Kits
• TUNR Endogenous kits
• Stably integrated, endogenous fine-tuned
knockdown
• Uses CRISPR-Cas9 technology
• TUNR AAVS1 kits
• Targeted insertion into AAVS1 safe harbor locus
• Uses CRISPR-Cas9 technology
• TUNR Transgenic kits
• Exogenous TUNR expression of your gene of
interest
• Faster results

13. Genetic Engineering DIY or Full Service

14. Cell line evaluation
(growth, clonability,
transfection, HR rate)
Transfection with CRISPR
NGS the pool
Single cell clone
Clonal genotyping by NGS
Clonal expansion
Mycoplasma test and
banking
Cell Line Project Example

15. Process
• Complete the
Evaluation Form:
• Applications
• Validation
• Service Offering
https://canopybiosciences.com/custom-cell-line-
engineering-2/
Turnkey solutions for genetic engineering

16. Knock In
From $19,900
Specifics of each project need
to be examined to determine
price

17. Complete custom cell line engineering
• Knockout cell lines in as little as 4 months
• Experience in dozens of tumor cell lines, iPSCs, ESCs
• Knockouts, knock-ins, TUNR knock-ins, point
mutations, gene tagging, exon swapping
• Milestone pricing
• Careful project management
• Designed to be affordable

18. Focus: Whole Genome Editing/Whole Genome
Monitoring
• Provide cutting edge tools and services for modifications to genes – any edit,
any genome
• Build technologies for whole genome monitoring and support them with the
highest level of service

19. nCounter: The Only Direct, Digital, Nucleic Acid-
Counting Technology
Single molecule fluorescent barcodes,
each attached to an individual nucleic acid molecule

20. Barcoded probes find and label their specific targets . . .

21. . . . each target is labelled with a specific
probe with a specific barcode . . .

22. . . . barcodes are scanned and counted

23. NanoString measures up to 800 mRNAs or miRNAs
(some DNA and protein, too)
• Small input needed (100ng)
• No amplification
• No optimization of individual probe
sets
• Works with degraded mRNA
(FFPEs)
• Medium throughput and quantitative
for approximately 40 cents per data
point

24. No Amplification
• Save on time
• Save on money
• No enzymes
• No polymerases
• No reverse transcription
• No PCR
• No library prep
• No library amplification

25. Nanostring Correlates with qPCR
99% C.I
Khan, et al. (2011)

26. Highly Reproducible: 3 Labs, 3 Countries,
Same Results
Rapid, Reliable, and Reproducible Molecular Sub-
grouping of Clinical Medulloblastoma Samples
Northcott P.E. et al., Acta Neuropathologica;
November 16, 2011
R2 Site1 v Site 2 = 0.97
R2 Site1 v Site 3 = 0.98

27. • Determining the cell-of-origin is
important for determining
treatment course for diffuse large
B-cell lymphoma (DLBCL)
• Nanostring gene expression
assay (20 genes) used to
determine cell-of-origin
• Assay accurately assigned
DLBCL subtypes
Classification
Prognosis
Validate MicroArray/Replace MicroArray

28. • In addition to standard kits (human, mouse, rat) customized panels
can be created for any genome
• 4-6 week turn around time for all customized panels
• Each customized panel can measure up to 800 genes
Customize NanoString for Any Genome

29. Gene Panels (mRNA/miRNA)
Kit Human Mouse Rat Genes
PanCancer Pathways ✓ ✓ 770
PanCancer Immune Profiling ✓ ✓ 770
PanCancer Progression ✓ 770
Immunology ✓ ✓ 594/561
Kinase ✓ 536
Inflammation ✓ ✓ 255/254
Cancer Reference ✓ 233
Breast Cancer ER ✓ 202
Stem Cell ✓ 199
Adaptive Immunity ✓ 192
Innate Immunity ✓ 192
Cellular Profiling ✓ 192
Cancer Metabolism ✓ 192
RNA-DNA Damage and Repair ✓ 192
Wnt Pathways ✓ 192
Cellular Signaling ✓ 192
Reference ✓ 18
miRNA ✓ ✓ ✓ 800/600/400 Custom
panels also
available

30. Canopy Offering: Service
Sample Type
• Format
• Number of samples
• Species
Gene Panel
• Predesigned or
custom panel
Data Analysis
• Level of analysis by
Canopy
1 2 3
Isolated RNA
Cells
Tissues
Tumors
FFPE
Whole blood
OTS – 2 days
Custom – 2 to 3 weeks

31. Exclusive Partner Nordic
Cobo Scientific
Jens-Ole Bock, M.Sc.
info@coboscientific.com
+45 24207221
www.coboscientific.com