Transposagen Genome Engineering Brochure 2014

Find Your Place in Genome Engineering

Transposagen Biopharmaceuticals, Inc. [email protected]

www.transposagenbio.com

Technologies, Tools & Services Brochure

piggyBac™ • XTN™ TALENs • NextGEN™ CRISPR • Footprint-Free™ Gene Editing • Custom Vectors • Cell Line Engineering • Custom Animal Models and More!

Connect with us for the latest updates on gene editing technologies, products, services and promotions!

Transposagen Biopharmaceuticals, Inc. www.transposagenbio.com

About Us

Welcome to Transposagen Biopharmaceuticals, Inc., a worldwide leader in genome engineering technologies and services with applications in therapeutics, research & drug discovery, bioproduction, clinical genetic testing and agriculture. Our products and services include Footprint-Free™ Gene Editing, NextGEN™ CRISPR, XTN™ TALENs, and custom cell lines, stem cells, and animal models. Our unique genome engineering capabilities allow for the creation of nearly any genetic modification in any genome.

• Anyone can cut DNA (even site-specifically)

• Our technology is the cleanestgene editing technology in the world

• We are the only company in the world that can edit as little as a single nucleotide without any unwanted changes and with the ability to select for rare events

• We have a 20-year patent position protecting this technology

Custom reagents and technology portfolio

Cell and animal model engineering services

Drug development partnerships

What Differentiates Us What We Offer

• Drug Discovery & Development Elucidating gene function & regulation Target discoveryDisease modelsToxicology & reporter lines

Isogenic control lines & diagnostics

• BioproductionHost engineering for improved production and function Stable cell lines and pools

• TherapeuticsGene therapyCell therapy – ex vivo cell therapy

Why Genome Engineering

[email protected]

The piggyBac™DNA Modification System


Transposagen is proud to provide the industry’s most efficient non-viral stable transfection & transgenesis technology. Choose any of our off-the-shelf transposons and stable transfection kits or custom transposon services in order to facilitate stable transfection and transgenesis. Simple, one step protocol from transfection to stable cell lines.

How It Works

Step 1. Clone your custom cargo into piggyBac™ transposon vector.

Step 2. Transfect the piggyBac transposon containing inserted cargo along with the piggyBac™ transposase (plasmid DNA or mRNA) into target cell.

Step 3. piggyBac™ transposase recognizes PB transposon sequences (ITR’s), cuts transposon, carrying the cargo out of the plasmid and integrates it stably in the genome.

Advantages No size limit- up to 250 kb has been published 80-100% stable integration efficiency Effective on all mammalian genome Precise Excision Non- Toxic and non-mutagenic

Applications• Stable cell line creation• Stem cell research: reprogramming, differentiation

and selection. • Protein production • Cell and gene therapy • Drug Screens • RNAi: inducible and reversible knockdown in cells and

animals models

Using piggyBac™ for Stable Transfection & Transgenesis

PB- GOLD™ Stable Transfection Kit Contents

• hyperactive super piggyBac™transposase (sPBo) plasmid

• lipofection reagents • positive control plasmid

Unmatched Stable Expression THP-1 Reporter Lines

Red = negative controlGreen = initial transfection expressionBlue = expression after 10 months

Mossine et al. 2013

Outperforms Viral & Plasmid Methods

[email protected]


piggyBac™ Excision-only TransposasepiggyBac™ excision only transposase allows you to remove cargo from the in a Footprint-Free™, seamless or scarless manner. Use for:

Footprint-Free Gene Editing Phenotype Reversion Studies

Custom Services Transposagen provides everything from vector to full cell line or animal model creation. • Custom Transposon vectors • Custom Cell Lines • Custom Transgenic animal models

piggyBac™ Footprint-Free™ Excision

PB-x GOLD™ Excision-only Transfection Kit Contents

• Excision-only piggyBac™ transposase (PBx) plasmid

• lipofection reagents • positive control plasmid

Rapid Transgenesis

Pettitt et al 2013

Genetic Screen’s & Phenotype Reversion Studies

Demonstration of the piggyBac™ capability to produce large stable mutant libraries in forward genetic gene trapping screens. Genes involved in compound (Olaparib)toxicity resistance were discovered (left). Followed by phenotype reversion back to sensitivity with excision only piggyBac™ (right)

Green = wild type sensitive cellsBlue = resistant cells with PB gene trap insertions

Grey = reverted excised clonesBlue= resistant cells with gene trap insertionGreen = wild type sensitive cellsRed = negative control

Off-the-shelf vectors Transposon backbone vectors for cloning your cargo of interest. The piggyBac™ ITRs flanking core insulators and a multiple cloning site (MCS). Our backbones come with a variety of promoters, markers and other features.

[email protected]

Site-Specific Nucleases for Gene Editing


Site-specific nucleases enable researchers to accurately cut any DNA sequence, allowing for the creation ofgene knock-outs, knock-ins and single base pair edits. Transposagen offers the widest variety of nucleasesincluding our XTN™ TALENs and the latest dimeric RNA-guided NextGEN™ CRISPR. Not only can Transposagenprovide you with the latest DNA editing technologies, but we have the fastest delivery time in the industry.

Genome Modification Applications

Avoid Off-Targets With NextGEN™ CRISPR

Some nucleases, especially the original CRISPR/Cas9technology have the unfortunate consequence of off-target mutations. The NextGEN™ CRISPR technology is thefirst truly dimeric RNA-guided FokI nuclease whichdiminishes these concerns. The NextGEN™ CRISPR systemempowers researchers to engineer the genome with thehighest fidelity without compromising the budget.

Affordable

Fast

High efficiency

Able to multiplex

Highest fidelity; no detectable off-

targets by deep sequencing

methods

• Gene knock-out and knock-ins• Single base-pair gene editing• Mutation correction or creation• Cell line & animal model engineering• Validation of GWAS studies and patient

clinical trial selection• Cellular therapies

NextGEN™ architecture. The NextGEN™ CRISPR utilizes the dimerization requirement of FokI for improved specificity.

By introducing site-specific double-strand breaks, nucleases promote the NHEJ and HR pathways which enable gene editing.

Custom Reagents

[email protected]

Succeed With XTN™ TALENs


Transposagen is an experienced XTN™ TALEN producer. In fact, we use XTN™ TALENs for our own in-houseprojects for producing genetically engineered rats and cells lines. Our XTN™ TALENs have to be of the highestquality and activity in order to achieve results for our customers. We deliver our commercial XTN™ TALENs withthe same standards in order to help you achieve your gene editing goals.

Get the best – Not all TALENs are equal. Transposagen uses the superior protein engineering to deliver high performance TALENs. Enhanced targeting, better promoters, variable repeats and codon optimization make all the difference. Have them fast – We utilize our own XTN™ high-throughput production system to deliver your TALENs quickly.Target your gene more often – XTN™ TALENs have a wide targeting range in the genome making all types of research possible.Acquire results – Higher efficiencies translate to better results.Stay out of trouble – XTN™ TALENS have minimal off-target effects and low toxicity.

Site-Specific Nuclease Product & Service OptionsTransposagen has a number of innovative site-specific nuclease service programs designed to fit the needs and budget

of any research or drug discovery project. We offer everything from off-the-shelf gene editing tools to the most complex animal model creation services.

Advantages of XTN™ TALENs

→ Value™ Program – Receive custom NextGEN™ CRISPR and/or XTN™ TALEN plasmids sequenced verified and transfection ready.

→ Guaranteed Success™ Program – Two custom NextGEN™ CRISPR and/or XTN™ TALEN plasmids sequenced verified and transfection ready. If the first pair of nucleases do not cut at your target you build more for FREE.

→ Gene Editing Kit – Two custom NextGEN™ CRISPR and/or XTN™ TALEN plasmids and custom donor vector sequenced verified and transfection ready.

→ mRNA Production Service – High quality mRNA production for any custom NextGEN™ CRISPR and/or XTN™ TALEN.

Take Your Nucleases To The Next LevelAsk your local Gene Editing Specialist about our unique Footprint-Free™ Gene Editing piggyBac™ reagents and services.

Combine NextGEN™ CRISPR With XTN™ TALENsUnlike other providers in the industry, Transposagen offers both CRISPR and TALEN technologies to researchers. Sinceeach nuclease has inherent sequence requirements, the option of both provides a wider targeting range than alone.Combine both nucleases in a single transfection for even higher chances of successful gene targeting!

Custom Reagents

XTN™ TALEN Rat Rosa26 targeting compared to a competitor TALEN producer.

XTN™ Competitor

Bands indicate activity by Cel1 assay

Evidence of XTN™ Efficiencies

• Hundreds of successful projects in cell line and animal model creation.

• Direct comparisons between our XTN™ TALENs and competitor producers.

[email protected]

Footprint-Free™ Gene Editing


Transposagen’s Footprint-Free™ gene editing kits & services combine the industry’s most precise, efficient, and flexible site-specific nucleases, NextGEN™ CRISPRs and XTN™ TALENs, with our exclusive piggyBac™ transposon system - the only commercially available method capable of seamless excision of resistance or reporter genes.

Footprint-Free™ Gene Editing allows users to increase efficiencies by utilizing selection enrichment for gene edits, followed by “footprint-free” or “scarless” removal of the selection cassette, resulting in clean genome editing.

How It Works

1- Start with the most efficient & precise targeted nucleases, NextGEN™ CRISPR and/or XTN™ TALENs to stimulate homologous recombination at the target site.

2- Use a Footprint-Free™ Gene Editing donor plasmid from our Multivector™ portfolio for targeted integration and selection enrichment of the desired gene edit.

3- Following selection Excision Only piggyBac™ (PBx) removes markers, leaving behind a scarless correctly edited genomic DNA.

Genome Modification Applications

• Gene editing down to a single base-pair –mutation correction or creation in cell lines with key advantages in difficult to transfect cells such as stem cells (iPS and ES cells) and primary cells.

• Targeted knock-ins, reporter lines and humanizations.

• Targeted knockouts and knockout reversions –introduce a disruption (selection marker) in the coding region, following phenotypic analysis remove the disruption Footprint-Free™ for phenotype reversion studies.

Product & Service Options Transposagen provides a range of services from the off-the-shelf Multivector™ portfolio to custom gene editing kits. Footprint-Free™ cell lines and animal model services are also available.

Multivector™ Catalog #

PB-MV1Puro-TK

PB-MV1Neo-TK

PB-MV1CMV-GFP-Puro

PB-MV1Neo

PB-MV1Hygro-TK

Technology Applications

[email protected]

Project Applications


Cell line engineering - Access to cell lines and stem cells from genetic diseaseor modified backgrounds is becoming more common and important for in vitro(cell based) modeling. Genomes vary in population, including SNPs in codingregions and regulatory elements as well as copy number. When compared to“control or regular stem cells” these natural variations result inmisinterpretation of in vitro phenotypes, making creation of isogenic lines usedas a reference for genetically modified or disease lines necessary for qualityresults.

Advantages in Stem Cells - Genome engineering in common transfection cell

lines such as HEK293 occurs at high efficiencies. However, stem cells are less

amendable to genome modification due to reduced transfection efficiencies

and potentially different DNA repair responses. Therefore, selection for

genome modifications through Footprint-Free™ Gene Editing is a key benefit

for stem cell engineering.

Cellular therapies – treatments for a broad range of disorders with clinicalcapabilities (no off-target mutations). piggyBac™ and Footprint-Free™ GeneEditing have been used in CAR T-cell therapy studies as well as correction ofmultiple disease mutations.

Isogenic cell line

creation

GWAS Study

Validation

Target discovery &

validation

Patient clinical trial

selection

Cell & Gene Therpay

Animal model

creation

Selection vs. single-stranded DNA oligonucleotide(ssODN) mediated gene editing: One alternative toselection is the use of ssODNs which are typically 60-120bases with the donor substitution (base-pair edits)flanked by homologous sequences on each side.Inefficiencies require a large number of clones to bescreened resulting in project timeline delays and costincreases. Footprint-Free™ selection enrichment allowssimpler easier screening, for faster and more costeffective genome editing.

Advantages of Footprint-Free™ Gene Editing

Why selection? Site-specific nucleases increase the efficiency of targeted knock-ins or gene edits significantly, butin many cases efficiencies may not be high enough for clone isolation. Therefore, the ability to select for desiredmutations and rare events is important for efficient gene editing.

Footprint-Free™ Gene Editing vs. CRE/loxPrecombinase: Recombinase methods remove drugselection markers from the genome by catalyzingrecombination between target sites (known as loxPsites). However, this process is not clean and leaves“footprints” or “scars” in the genome which havebeen shown to disturb splicing elements oralter/silence gene expression. In addition,recombinases recognize “cryptic” loxP sites andproduce off-target mutations, compromising results.Footprint-Free™ gene editing does not have theseadverse effects providing precise and efficientgenome editing for quality results.

Access to Footprint-Free™

Gene Editing1- Multivector™ Portfolio – generate your own Footprint-Free™ gene editing vectors

2- Custom Footprint-Free™ Gene Editing Kits – Let us design and produce the vectors for you

3- Custom Cell Line Engineering & Animal Model Creation – Utilize our expertise and outsource the whole project

Technology Applications

[email protected]

Custom Cell Line Engineering


Transposagen utilizes expertise and a diverse portfolio of gene editing tools including XTN™ TALENs, NextGEN™ CRISPRs, piggyBac™ transposons and Footprint-Free™ Gene Editing to deliver high quality custom engineered cell lines

Available Services Stable cell lines (transgenic,

overexpression, inducible) Knockout Knock-in (humanization &

conditional) Footprint-Free™ Gene Editing Cell line characterization services

Custom Services

An example of our 4-step milestone pricing structure for high efficiency knockout cell lines

Best ReagentsYour custom engineered cell line begins with the latest gene editing reagents available. Transposagen uses TALEN and CRISPR technologies, including the latest dimeric NextGEN™ CRISPR which has been shown to have minimal off-target effects. Combined with piggyBac Footprint-Free Gene Editing, Transposagen achieves the highest efficiency.

ExpertiseTransposagen has over 10 years of experience engineering custom cell lines and animal models. We've successfully delivered projects to the top 20 pharmaceutical companies and all the major academic institutes. We have experience with many cell lines including iPS, SSCs, CHO, HEK293, HeLa, ES, SH-SY5Y, HepG2, A549, MCF7, CACO-2, RAW264.7, THP-1, Jurkat... to name a few!

TimeGenerating a cell line can be time-consuming and complex. By utilizing the piggyBac™ system, Transposagen can delivery engineered cell lines faster than anyone in the industry for even the most difficult lines.

Accurate Pricing & Flexible Projects No surprises. Prior to the start of any project, Transposagen can accurately price out the cost of your engineered cell line so you know what to expect. All cell line projects are divided into milestone payments which allows researchers to start the service without paying for all of it upfront. You only pay for the work we complete!

Keep Your InvestmentThe reagents we use to engineer your cell line are yours to keep. Use them again in a different cell line or take them to our animal model services to maximize your research efforts.A demonstration of knockout

cell line creation at Transposagen. Inactivating the fucosylation system in CHO cells for producing afucosylated human therapeutics. Antibodies expressed from these host cells were afucosylated and demonstrated the expected increased ADCC activity. In collaboration with AragenBioscience.

Targeting Fut8, the alpha1,6-fucosyltransferase gene

with XTN™ TALENs in CHO Cells

[email protected]

Custom Animal Models


Transposagen has the industry’s most versatile collection of genome engineering technologies as well as expertise in rodent pronuclear injection (PNI) and stem cell engineering. These capabilities result in the most

flexible and robust custom model generation services in the industry. The more targeted and complicated your therapeutics become, the more you need the right partner to advance your research. Working intimately with

our scientific team will help you bring innovative solutions and products to market faster.

Custom Services

Available Rat & Mouse Services Knockout Transgenic Overexpression Conditional Knock-in Transgenic knockdown Humanized Disease Models

[email protected]

Genome Engineering Vector Design & Validation+

PNI, ES or SSCs

High-speed, high-efficiency genome engineering

Rodent Model Creation Strategies

Method Species Targeted Knock-in Limitation

Estimated Timeline

Pronuclear Injection (PNI) Mouse & Rat ~2kb 4 months

Embryonic Stem Cells (ES) Mouse None 8 months

Spermatogonial Stem Cells (SSC)

Rat None 8 months

• Custom Engineered Rodents in as little as 4 months• Spermatogonial Stem Cell (SSC) technology is the only commercial method for producing

large targeted knock-ins in rats


FatRat™ Mc4r Knockout Obesity-Diabetes Model

Research Models

Featured Knockout Rat ModelsDrug Metabolism• BcrpImmune System• Rag2 - SCIDNeuroscience –CNS• SERT• OPRL1• Ghsr• Nrg1• Trpc4• Pde4d• Myo9aMetabolic Disease/Obesity• Mc4r – FatRat™• LeptinCardiovascular• Sod3Cancer/Toxicology• P53

[email protected]

Transposagen’s off the shelf TKO® and TGEM® rat models cover a range of disease and research applications. We have partnered with world experts in over 40 collaborations worldwide in order to validate and characterize our genetically engineered rat models. We have approximately 300 off-the-shelf models which is the world’s largest commercially available collection. Our characterized genetically engineered models are broken down into the

research categories below.

• Most common monogenic obesity gene• Obesity studies, including early and late onset obesity• Diabetes research• Metabolism studies• Renal studies• Altered Leptin Signaling Pathway

Rag2 –SCID Knockout Rat

• Cell sorting data demonstrates no mature T and B cells

• Western blot /Immunochemistry indicates no IgG

• Initial results indicate that the animals are athymic

New SCID Rat for Human Xenograft Tumor Models

Access to Research Models

Unlimited breeding licenses - breeding at the customer’s in-house facility or a contract breeder of choice. The license comes with at least 2-3 heterozygous breeding pairs.

Trial licenses- allow the customer to purchase a limited breeding license to conduct pilot studies at a lower purchase price.

Individual animals – may be available, please inquire

Transposagen Biopharmaceuticals, Inc. [email protected]

www.transposagenbio.com

Connect with us for the latest updates on gene editing technologies, products, services and promotions!

Exon Exon Exon

One-Step Stable

Transfection & Transgenesis Targeted Knockouts

Footprint-Free™

Gene EditingNextGEN™ CRISPR

Stable Transfection Kits &

piggyBac™ TransposonXTN™ TALEN

Knock-ins &Humanization

Gene Editing

Base-Pair Changes

Cell Line & Rodent Model Engineering Services

Your Next Discovery Awaits

Targeted Selection

Markers

With So Many Opportunities

What Are You Waiting For?

Next-Generation Genome Engineering

Documents

Transposagen Genome Engineering Brochure 2014