leap-in transposase(R)Efficient multi-site integration

Genetic stability

Unlimited payload

Faster cell line development

Application-optimized expression constructs

Shortened stable pool recovery times

Stable integration combined with structural integrity

Maximize expression levels using ATUM’s proprietary gene sequence and vector optimization tools.

Clonal productivity distribution is characteristically higher and more uniform in stable pools.

Transposases integrate the entire, intact transposon, thereby maintaining its structural integrity, and expression balance.

Adjust and tune expression levels of your gene(s) by testing combinations of vector elements.

Fewer clones need to be screened to enable identification of highly productive clonal cell lines.

Clonal cell lines created using Leap-In transposases exhibit genetic stability and stable productivity over 60 population doublings.

Express multichain proteins (such as bispecific antibodies) or multiple genes within a pathway.

Initiate process development earlier with more representative pool derived reagents.

Control integration copy number by manipulating dose of transposase and synthetic transposon. Footprint-free excision of transposon from host genome.

Leap-in Transposase(R) Benefits

• Integration sites are enriched in transcriptionally active chromatin.

• Multiple copies of a transposon are independently integrated into the genome of a single cell.

• Transposition can be achieved in a broad range of hosts.

• Transposon excision perfectly restores the original genomic sequence.

• The entire sequence between two transposon ends is integrated into a host genome, and there is no payload size limit.

• Transposon does not jump in the absence of cognate transposase.

• Multiple orthogonal transposases are available.

Alternative to AAV and Lentivirus gene delivery systems.

Background

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Multiple integrationsby cut & paste

Transfect

+ Transposase Inverted Terminal RepeatExpression Construct

Transposon Recognition Site

Transposase protein

legend

ATUM has developed a set of tools for genomic integration of DNA constructs based on novel transposons and their cognate transposases. This system features several properties that make it particularly well suited to engineering mammalian cell lines for bioproduction:

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Leap-in Transposase(R)

The main limitation in creating cell lines from multi-ORF constructs is what happens to the DNA when it gets into the cell. Typically DNA is randomly fragmented and those fragments are integrated with additional rearrangements and concatemerization which may limit the benefits of providing the genes in a single construct.

Structural integrity of the complete expression cassette is maintained in the presence of transposase as shown in the left panel. In the absence of transposase (right panel), structural integrity of the expression cassette is compromised as seen by

truncations plus random concatemerization and scrambling of the expression cassette. HC: Heavy Chain, LC: Light Chain, ITR: Inverted Terminal Repeat, GS: Glutamine synthetase.

Leap-in transposases integrate the entire transposon into the expression host genome, thereby maintaining the structural integrity of the construct, consistent linkage of all genes, and the desired expression ratio between protein chains.

LC LCGS GSHC

+ Transposasemultiple independent integrationsof complete expression cassette

Designed Construct

- Transposasetruncations of expression cassette

plus random concatemerization and scrambling

GS ITRITR HC LC

GS ITRITR HC LC

GS ITRITR HC LC

GS ITRITR HC LC

GS ITRITR HC LC

GS ITRITR HC LC

GS ITRITR HC LC

GSHC LC

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Maintaining Structural Integrity

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Antibody Expression

TRANSPOSASE

The Leap-in transposase integrates all DNA between the two transposon ends. The selectable marker and all encoded open reading frames are therefore 100% linked. This is a very effective way to integrate genes that need to be expressed together, for example the heavy and light chains from monoclonal antibodies or the three or four chains of bispecific antibodies.

The relative expression levels of the different chains of an antibody often have a profound effect on the amount of antibody that can be produced. By using well characterized regulatory elements, ATUM can ensure optimal ratios of expression between different open reading frames within a construct. Multiple copies of a transposon within the genome arise from independent integrations at different sites. These are more stable than the concatamers that arise from random integration. Independently integrated transposons are also not susceptible to repeat-induced silencing.

Transposase Speeds Recovery and Allows Increased Selective Pressure

Transposase speeds recovery. CHO K1 GS KO cell line from Horizon Discovery (catalog# HD-BIOP3) was co-transfected with transposon and transposase as shown. A. Under drug-free selection conditions (attenuated GS promoter), cell pools created using transposase (curve 1) recover viability in ~3 weeks, whereas cell pools created without transposase (curve 2) fail to recover. Curve 3 is the mock negative control. B. Attenuated GS cassettes recovered in the presence of 15mM MSX (curve 4), or 5mM MSX (curve 5). No recovery was seen when transposase was not used (curve 6).

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Although the Leap-in transposases share many desirable features of the piggyBac transposase from the looper moth Trichoplusia ni, they share only about 20% sequence identity and fall outside patent claims for the looper moth system. Five US patents protecting the Leap-in system have already issued (9,428,767, 9,534,234, 9,574,209, 9,580,697 and 10,041,077), with many more applications pending in the US and abroad.

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Transposons with substantially attenuated selectable marker expression cassettes are integrated by transposases. When selectable marker expression from a single cassette is very low, recovery requires multiple copies to be integrated at sites in the genome favorable for gene expression. The combination of transposases and transposons with attenuated selectable marker cassettes thus produces highly productive pools of cells. Sample pool productivities are shown in the table below.

HCGS

promotervolumetric

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productivity

IgG1-Hs ht 4.2 g/l 42 pcd

IgG1-Hs ht 3.6 g/l 29 pcd

IgG1-Hs ht 3.3 g/l 29 pcd

IgG1-Hs ht 2.8 g/l 30 pcd

IgG1-Hs hxt 4.2 g/l 33 pcd

IgG4-Hs hxt 5.0 g/l 43 pcd

IgG4-Hs hxt 5.0 g/l 49 pcd

Examples of Stable Pool Productivity

Cells are grown under selective conditions to produce stable pools. These are ranked by productivity and product quality. At this point material can be generated from the stable pools and single clone isolation is initiated.

HD-BIOP3 GS null CHOK1 cell line from Horizon Discovery was co-transfected with ATUM’s transposase and transposons with different vector and antibody combinations. Stable pools were established and productivity measured in non-optimized small scale shake flask or deep-well cultures.

Examples of Stable Pool Productivity for Different Molecule Types

proteinselection

stringencytransposon

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300 kDa glycoprotein-Fc fusion low 5 to 7 ~4bispecific antibody medium ~16 7 to 12

antibody medium 20 to 25 35 to 50antibody high 35 to 45 50 to 70

Productivity and copy number is maintained after 60 population doublings.

Stability of Stable Pools

Productivity Maintained Copy Number Maintained

Graphs show the maintenance of productivity and transposon copy number in clonal cell lines after 60 population doublings (PD). A subset of data taken from 47 clones created using Leap-In technologies is shown. All clones retained at least 70% of their initial productivity and transposon copy number.

Leap-in transposase protein acts to integrate the transposon into the host genome. Because each integration contains a single copy of the transposon, there is no instability resulting from concatemer recombination or repeat-induced silencing.

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www.atum.bio TRANSPOSONS AND TRANSPOSASES

pD2500 & pD3600 Stable Vectors

ATUM offers two stable vector series: the pD2500s and pD3600s, which share a similar structure. The pD2500s are compatible with one of our Leap-in transposases (LPN-1), but they do not require a transposase for good levels of expression. The pD3600s are compatible with both of our Leap-In transposases (LPN-1 and LPN-2), and we recommend that they only be used with a transposase. This is because we have deliberately attenuated the selectable markers in the pD3600s, so multiple integration events are required for cell viability under selection. The pD3600s are therefore capable of higher expression yields.

The pD2500 and pD3600 vectors are constructed in a modular way that allows easy modification of component elements. Versions are available that allow expression of up to four open reading frames in addition to the selectable marker.

Our stable vectors are built from several sets of mammalian functional elements:

⬤ Metabolic selectable markersGlutamine synthase (GS), dihydrofolate reductase (DHFR)

⬤ Drug-resistance markerspuromycin, blasticidin, hygromycin, neomycin, zeocin

⬤ PromotersEF1a, CMV (murine and human), hybrids

⬤ RNA processing elementsIntrons, post-transcriptional response elements

⬤ InsulatorsFlanking the construct, and isolating transcriptional units from each other

The total number of possible combinations of vector elements is very large, and ATUM does not have all of these built. However construction is modular, so it is easy for us to create custom combinations if we don’t have the combination you want.

Promoter 1

ORF1 signal

ORF1

ORF1 poly A

intervening sequence

Promoter 2

ORF2 signalORF2

ORF2 polyAInsulator

Transposon right border

Bacterial Origin

M_Kanamycin-r_*

P kanamycin

Insulator

Marker polyA

Mammalian markerMarker promoter

Leap-In Transposon

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One Vector, Two Open Reading Frames

Co-transfection of separate vectors containing genes for heavy and light chains is a common method for producing antibody from transiently transfected cells. This approach is less desirable for stable cell line generation. The production of properly assembled antibody requires sufficient light chain to help the heavy chain fold. However a great excess of light chain diverts cellular resources away from producing enough heavy chain to obtain high titers. If genes encoding the two chains are incorporated into the production host genome independently, the number of gene copies integrated, and integration position effects, will influence the relative expression of the two chains. This results in a higher downstream screening burden during the cell line development process.

A preferable method is to express both genes from the same construct. In this case regulatory elements can be chosen to produce a desired expression ratio between the two chains. The table below shows 9 different construct configurations. The total productivity differs by less than 30% for any of the constructs, but ratios of between 1:1 and 3:1 can be achieved. We find that this ratio is influenced by the 3´ sequences of the first gene, as well as the promoter sequences driving the second.

gene ID first promoterfirst

polyAsecond

promoter chain ratio productivity heavy chain

269998 EF1a-Hs a A 2.96 24,164 6,126

269986 EF1a-Hs a B 2.86 24,442 6,339

269996 EF1a-Hs a C 2.53 30,799 8,608

270003 EF1a-Hs b D 1.97 24,182 7,846

270005 EF1a-Hs c C 1.81 33,991 12,129

270000 EF1a-Hs d E 1.78 23,058 8,037

269984 EF1a-Hs b C 1.17 25,303 11,699

269976 EF1a-Hs d F 1.10 32,845 15,887

270006 EF1a-Hs e B 1.01 30,603 15,548

Genes encoding DasherGFP and CayenneRFP were cloned into pD3641h in 9 different dual-promoter configurations as shown above. Constructs were transfected with LPN-1 transposase mRNA into CHO-K1 GS KO cells, and selected in media without glutamine. After recovery, DasherGFP and CayenneRFP fluorescence were measured and used to calculate the relative expression of the two

proteins (in arbitrary units). The total fluorescent protein expression is shown in the “productivity” column. Expression of the lower expressing proteins is shown in the “heavy chain” column. The ratio of expression of the two proteins is shown in the “chain ratio” column.

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Two identical ORFs coding for a secreted protein were tagged with V5 (ORF1) or FLAG (ORF2). A combination of vector elements were utilized to control the ratio of expression of expression of ORF2 to ORF1. Expression ratios of ORF2 : ORF1 between 2.5 : 1 and 1 : 1.5 were demonstrated.

Similar combinations of control elements can be used to create constructs carrying 3 or 4 open reading frames, enabling expression of all of the chains for a bispecific at controlled ratios.

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TRANSPOSASE AND CELL LINE DEVELOPMENT

Left transposon endMarker polyA

M_Glutamine synthetase_*

Promoter for Marker

ORF1 Promoter

ORF1

ORF1 polyAORF2 Promoter

ORF2

ORF2 polyA

ORF3 Promoter

ORF3

ORF3 polyA

Right transposon end

pUC oriKanamycin R

kan promoter

3 Slot Co-linear12373bp

Left transposon endInsulator

Marker polyA

Mammalian marker

Promoter for Marker

ORF1 polyA

ORF1

ORF1 Promoter

ORF2 polyA

ORF2

ORF 2 PromoterSpacerORF3 promoter

ORF3

ORF3 polyA

ORF4 Promoter

ORF4

ORF4 polyA

Insulator

Right transposon end

pUC ori Kanamycin RKan promoter

4 Slot Divergent18357bp

Combinations of control elements can be used to create constructs carrying 3 or 4 open reading frames, enabling expression of all of the chains for a bispecific at controlled ratios.

Constructs with 3 open reading frames (ORFs) in a co-linear configuration with combinations of control elements enables expression of all 3 ORFs at controlled ratios.

Constructs with 4 open reading frames (ORFs) in a divergent configuration with combinations of control elements enables expression of all 4 ORFs at controlled ratios.

Genes encoding fluorescent proteins were cloned into pD3641h in 3 or 4 slot co-linear or divergent multi-promoter configurations as shown above and page 8. Constructs were transfected with LPN-1 transposase mRNA into CHO-K1 GS KO cells, and selected in media without glutamine. After recovery, fluorescence were measured and used to calculate the relative expression of the fluorescent proteins (in arbitrary units). The

ratio of expression of the 3 or 4 proteins is shown. For the 3-ORF vector, various ratios of all three ORFs were achieved. For the 4-ORF vector, a 4-fold range of expression was observed between the different ORFs. Transposase allows insertion of the entire expression cassette enabling expression of each ORF at controlled ratios.

One Vector, Three or Four Open Reading Frames

Examples of expression ratios in 3 and 4 open reading frame (ORF) constructs

Regulatory elements can be chosen to produce a desired expression ratio between the proteins. The graphs above show examples of expression ratios in constructs with 3 and 4 co-linear or divergent multi-promoter configurations. Similarly, different expression ratios can be achieved by combinations of control elements, enabling expression of all of the chains for a bispecific at controlled ratios.

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Leap-in transposase mRNA in cells is transient

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Monitoring Leap-in Transposase(R) ActivityGenetic stability is established by copy number, productivity and consistent growth rate. Hypothetic transposase activity acting on the stably integrated transposons could potentially induce instability and integration site change. We have demonstrated using TLA (Targeted Locus Amplification) based integration site mapping that there was no change in numbers and genomic positions of integrated trangenes over >100 population doublings.

Leap-in transposase mediates footprint-free excision

To charactertize (qualify) a host cell line, extended passage cultures can be monitored using a functional assay using a reporter construct utilizing the footprint-free excision feature of Leap-in transposases as shown.

A synthetic transposon (RFP gene flanked by Leap-in 1 ITR’s inside a GFP gene flanked by Leap-in 2 ITRs) was integrated into CHO cells using Leap-in 2 transposase. Cells exhibited red fluorescence as RFP is expressed.

Cells transfected with Leap-in mRNA. Leap-in 1 transposase excised RFP at cognate ITRs.

Cells exhibited green fluorescence as intact GFP is expressed

y = 1133.8e-0.057x

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NG GS KO CHO cells were transfected with LPN-1 mRNA, samples were taken from different time points post-transfection. Total RNA was extracted from NG GS KO CHO cells for RT ddPCR. FAM labeled primers targeted the LPN-1 RNA and HEX labeled primers for mouse actin used as positive control.

Excision Efficiency of Transposase

Readouts:• Green fluorescence upon RFP cassette

excision. • No green fluorescence detected after 3

months of cell culture.

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www.atum.bio TRANSPOSASE AND CELL LINE DEVELOPMENT

Contact us for information on licensing options & cell line development services

+1 650 853 8347 ext. 200+1 877 DNA TOGO ext. 200info@atum.bio

Flexible Licensing Options for Leap-in Transposase(R)

EvaluationTest and optimize the Leap-in technology in your labs. Convert to a full research license after 6 months.

ResearchAnnual license fee. Access to all new transposon and transposase technology developed at ATUM.

CommercializationPayable on cell lines that enable commercialization of your therapeutic, diagnostic or product. Pay annually, as a single lump sum, or attached to clinical milestones.

Bring the advantages of Leap-in technology to your in-house cell line development. We offer three tiers of licensing to meet your research and commercialization needs. Under all licenses, Leap-in mRNA and synthetic transposon DNA are purchased from ATUM. Genes and vectors designed and synthesized by ATUM.

Transfected Leap-in transposase mRNA in cells is transient and is below the detection limit (~0.1 molecules of transposase mRNA per cell) in ~150 hours.

Transfected Leap-in transposase DNA in cells is undetectable (detection limit is ~0.002 molecules of transposase DNA per cell) in established clones.

+1 877 DNA TOGO+1 650 853 8347info@atum.bio

research. create. break through.