GENOME ENGINEERING SERVICESFOR CELL THERAPY MANUFACTURING

GENOME ENGINEERING FOR CELLTHERAPY MANUFACTURING
HARNESS THE POWER OF CELL ENGINEERING FOR
YOUR THERAPY DEVELOPMENTGene engineering is a powerful tool for fine tuning cell phenotype
in immune cell and regenerative medicine therapies. It allows theintroduction of new genes into cells and very precise alteration oftheir existing genes. Cell engineering can augment cell therapy
effectiveness by increasing cell potency and persistence in vivo. It
has recently been used to:• Express proteins that specifically target tumor cells, e.g.
CAR-T cells, TCR• Suppress immune responses in autoimmune disease andchronic inflammation• Mask cell therapies to avoid host immunity• Enable cells to infiltrate the target tissue• Deliver genes and correct genetic defects for gene therapy

RAPID AND FLEXIBLE GENE DELIVERY WITH TCBUSTER
TcBuster is a non-viral gene delivery system that enables stable gene transfer into any cell type. It is based on a transposon from thered flour beetle. The TcBuster system includes a transposon plasmid containing the gene of interest (GOI) to be inserted in the targetcell’s genome.
TcBuster supports the rapid generation of transgenic mammalian cells by allowing for multigene transfer and CRISPR-mediated knockouts
in one operation. TcBuster is a valuable tool for both proof-of-concept studies and manufacturing scale process development.

TcBuster™
1. Transfection of TcBuster Platform

OVERVIEW OF TcBuster TRANSPOSITION MECHANISM
TcBuster Transposon
2.

TcBuster mRNA

1. TcBuster platform introduced into the cells via electroporation

3.

2. TcBuster mRNA translated into TcBuster transposase
3. TcBuster transposase binds to ITR regions on transposon plasmid

4.

4. TcBuster transposase CUTS gene of interest (GOI) from plasmid

TcBuster transposase

5. GOI INSERTED into cell's DNA
5.

6. GOI mRNA transcribed from cell's DNA
7. Translated protein from GOI mRNA is now stably expressed in cells

6.

7.

Stable expressionof protein

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THE RED FLOUR BEETLE (TRIBOLIUM CASTANEUM)
The TcBuster transposon is derived from this beetle that feeds onbroken grain kernels and flour in food storage areas. The beetle
is dark red or brown, and it can fly and live for over a year. For
the laboratory-minded, the red flour beetle is easy to maintainin culture. Over their lifetime, females may lay up to 1,000 eggs
which hatch in 5-12 days.

Learn about Genome Engineering Services | rndsystems.com/products/tcbuster-gene-transfer-system

HOW DOES THE TCBUSTER SYSTEM COMPARE TO VIRUS-BASED SYSTEMS?
While both these approaches are designed to deliver genetic material to a target cell, viral methods for genetic modification (e.g. withlentivirus or AAV) involve transduction of the target cells, while non-viral transposons are integrated into target cells using standardtransfection methods (e.g. electroporation). Both approaches provide a means of cell entry for the gene of interest (the cargo) anda mechanism for integrating the GOI into the cellular genome. The advantages of TcBuster for cell therapy manufacturing include:• Reducing the time required and the cost of introducing GOI• Increasing the practical GOI cargo capacity compared to virus-based methods• Avoiding inconsistent reagent availability

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TCBUSTER EDITS PRIMARY HUMAN T CELLS SIMILAR TO LENTIVIRUS

Primary human T cells were modified by introducing a CD19 CAR with TcBuster(left) and lentivirus (right). Flow cytometry analysis shows that TcBuster modified agreater percentage of the target cells and led to higher expression of the CAR
compared to lentivirus transduction.

Flow cytometry analysis of CCR7 CAR insertion following transposition withTcBuster (A, B) or transduction with lentivirus (C, D). Cells were gated for CD4 (A, C)
or CD8 (B, D) expression. TcBuster modified cells had a greater percentage ofCD45RA+ cells (naïve T cells) compared to lentivirus transduction.

Three genes were transposed into T cell cultures of 10 x 106, 80 x 106, or 400 x 106
cells. All genes were delivered in a single reaction. At all scales, transposition
efficiency remains above 30%.

CD19 CAR-T cells engineered with TcBuster (red and purple) or lentivirus (blue andbrown) show similar cytotoxicity against CD19-expressing Nalm6/luc cells. The
non-electroporated (EP) controls (black and green) do not kill cells.

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GENOMIC LOCUS INSERTION PREFERENCES

TcBuster inserts genes into the genome in a nearly random pattern. It shows a lower preference for active genes than seen with viral methods, and this translates to a
reduced possibility that transposition will disrupt critical cellular genes.

COMPLEX GENE EDITING WITH
TCBUSTERWith the TcBuster system, a one-step process can be used toengineer knockouts and TcBuster-mediated gene delivery without
sacrificing cell growth.
The transposition of a CD19-GFP CAR was successfully completed
in combination with knockout of beta 2-Microglobulin.

5ADDITIONAL PRODUCTS AND SERVICES FOR CELL THERAPYMANUFACTURING
To complement your genome engineering program, our comprehensive portfolio of high-quality products will enable you to develop
and rigorously characterize engineered cells. We also offer custom development and GMP manufacturing services so we can meetyour particular requirements.

REAGENTS AND ASSAYS• Flow Cytometry Reagents and Kits – over 15 fluorophores withthousands of antibodies• Fluorescent-Labeled Proteins – for ligand-based analysis ofCAR-transposed cells• RNAscope™ ISH – confirmation of genetic modification in intactcells and tissue biopsies• Immunoassays – multiple formats for quantitative, precise, andreproducible results• Custom Services for Cell and Gene Therapy including custom
vialing of GMP proteins, GMP small molecules, and GMP
antibodies

CELL MANUFACTURING• GMP Proteins – cytokines and growth factors for ex vivo use incell manufacturing processes• GMP Small Molecules – reprogramming stem cells forregenerative medicine processes• Cloudz™ Cell Activation Kits – GMP and RUO immune cell
expansion with dissolvable microspheres• GMP ProDots™ Proteins – pre-aliquoted, rapidly dissolvablecytokine microspheres for closed system cell culture• ExCellerate™ Media – xeno-free formulations for definedcomposition culture of immune cells

6ANALYTICAL INSTRUMENTS• iCE Maurice™ – automated capillary electrophoresis with pre-assembled cartridges and onboard sample mixing• Micro-Flow Imaging™ – analysis of protein aggregation and particulate contamination• Simple Plex™ – automated cartridge-based ELISAs with 4-5 log dynamic range• Simple Western™ – automated capillary-based immunoassays for size- and charge-based complex sample screening• Single-Cell Western™ – analysis of protein expression in ~1,000 single cells in parallel• Luminex® - for multiplex immunoassays to screen cell culture samples for secreted proteins

FOR APPLICATIONS OF TCBUSTER IN
IMMUNE CELL THERAPY
To complement your genome engineering program, ourcomprehensive portfolio of high-quality products will enable you
to develop and rigorously characterize engineered cells. We alsooffer custom development and GMP manufacturing services so
we can meet your particular requirements.

To learn more | scaleready.com
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WHAT CAN I EXPECT FROM GENOME ENGINEERING SERVICES?
Whether you are new to gene engineering, have limited experience, or represent a gene engineering core group with limited bandwidth,
we provide the same level of responsive partnership and customer service. We will connect scientist-to-scientist and walk through theentire project with you. For projects ready for transition to the clinic, we will discuss proof of concept projects and process development
work. For all projects, we will work with you to:• Identify the scope of the request• Define the best engineering and cell assays that align with yourproject• Refine the project specifics, milestones, and deliverables• Review a statement of work
• Deliver a timely quote• Deliver regular project updates• Deliver your customized products

OTHER CELL ENGINEERING SERVICES
CELL LINES AND PRIMARY CELLS
No cell line is too difficult for us to engineer, and our highly efficient genome editing process speeds up delivery timelines. Regardlessof the project, we can customize all deliverables including the number of cells/vial, validation testing, datasheet preparation, and batchrecord documentation.
• RUO, GMP, and xeno-free capabilities• Gene engineering with any platform for KO, KI, or multiplex edits• Correction or introduction of single nucleotide polymorphisms (SNPs)• Isolation of primary cells at small or large scale• Validation of engineered cells – genetic, protein expression, and custom functional analysis• Banking of wild-type clones and engineered cells

INDUCED PLURIPOTENT STEM CELLS
Induced pluripotent stem cells (iPSCs) are produced by the reprogramming of differentiated somatic cells such as PBMCs, fibroblasts,and patient-specific samples. They can be engineered to address genetic defects, promote the rebuilding of damaged tissues, andavoid immune rejection after implantation.
• iPSC reprogramming, expansion, and master cell banking – all performed in-house• Feeder, feeder-free, and xeno-free cell culture expertise• Customizable validation assays and documentation

Inquire about Genome Engineering Services | bio-techne.com/services/gene-engineering-services
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OUR CAPABILITIES FOR GMP MANUFACTURING
Our Contract Development and Manufacturing Organization (CDMO) services cover large scale isolation, cell engineering and activation,and final fill and finish. Our services include optimization of process cost and performance repeatability, dual sourcing of criticalcomponents, and incorporation of key flexibility points to avoid non-conformity.

• Prepare CAR-T, TCR, or iPSC processes for GMP manufacturing• Sample processing for Phase I and Phase II clinical trials• GMP manufacturing, culturing, and banking of iPSC cell lines

CLEAN ROOM FACILITIES• ISO Class 7 certification (Class 10,000) meeting U.S.
requirements• 2 cell processing suites• 6,000 sq. ft. GMP manufacturing space in Minneapolis, MN, USA• 2 development labs and one in-house quality control lab

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NOTES:

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WHERE SCIENCE
INTERSECTS INNOVATION ™

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