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Leap-In Transposase(R)

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Leap-In Transposase(R) leap-in transposase(R) Efficient multi-site integration Genetic stability Unlimited payload Faster cell line development Background 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: • Integration sites are enriched in transcriptionally active chromatin. • Multiple copies of a transposon are independently integrated into the legend genome of a single cell. Transposon Recognition Site • Transposition can be achieved in a + Transposase Inverted Terminal Repeat broad range of hosts. Expression Construct Transposase protein • Transposon excision perfectly Transfect restores the original genomic sequence. Multiple integrations by cut & paste • The entire sequence between two transposon ends is integrated into Nucleus Nucleus a host genome, and there is no Cell Cell Chromosome Chromosome payload size limit. • Transposon does not jump in the absence of cognate transposase. • Multiple orthogonal transposases Alternative to AAV and Lentivirus gene delivery systems. are available. Leap-in Transposase(R) Benefits Application-optimized Shortened stable pool recovery Stable integration combined expression constructs times with structural integrity Maximize expression levels Clonal productivity distribution Transposases integrate the using ATUM’s proprietary is characteristically higher and entire, intact transposon, thereby gene sequence and vector more uniform in stable pools. maintaining its structural optimization tools. integrity, and expression balance. Adjust and tune expression Fewer clones need to Clonal cell lines created using levels of your gene(s) by be screened to enable Leap-In transposases exhibit testing combinations of vector identification of highly genetic stability and stable elements. productive clonal cell lines. productivity over 60 population doublings. Express multichain proteins Initiate process development Control integration copy (such as bispecific antibodies) earlier with more representative number by manipulating dose or multiple genes within a pool derived reagents. of transposase and synthetic pathway. transposon. Footprint-free excision of transposon from host genome. Maintaining Structural Integrity 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. Designed Construct ITR HC LC GS ITR + Transposase - Transposase multiple independent integrations truncations of expression cassette of complete expression cassette plus random concatemerization and scrambling ITR HC LC GS ITR HC LC GS ITR HC LC GS ITR HC GS ITR HC LC GS ITR HC ITR HC LC GS ITR HC LC ITR HC LC GS ITR LC GS ITR HC LC GS ITR GS ITR HC LC GS ITR HC LC GS LC GS Structural integrity of the complete expression cassette is truncations plus random concatemerization and scrambling of maintained in the presence of transposase as shown in the left the expression cassette. HC: Heavy Chain, LC: Light Chain, ITR: panel. In the absence of transposase (right panel), structural Inverted Terminal Repeat, GS: Glutamine synthetase. integrity of the expression cassette is compromised as seen by 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. 2 201903 www.atum.bio TRANSPOSASE Antibody Expression 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. 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 A. B. 120 120 100 100 1 80 80 60 60 4 Viability Viability 5 40 40 2 20 20 3 6 0 0 0 1 7 14 19 23 26 0 5 9 17 20 Time (Day) Time (Day) 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). 3 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. 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. GS volumetric specific HC promoter productivity productivity IgG1-Hs ht 4.2 g/l 42 pcd IgG1-Hs ht 3.6 g/l 29 pcd HD-BIOP3 GS null CHOK1 cell line from Horizon Discovery was co-transfected with ATUM’s transposase IgG1-Hs ht 3.3 g/l 29 pcd and transposons with different vector and antibody IgG1-Hs ht 2.8 g/l 30 pcd combinations. Stable pools were established and productivity measured in non-optimized small scale IgG1-Hs hxt 4.2 g/l 33 pcd shake flask or deep-well cultures. IgG4-Hs hxt 5.0 g/l 43 pcd IgG4-Hs hxt 5.0 g/l 49 pcd Examples of Stable Pool Productivity for Different Molecule Types selection transposon specific protein stringency copy/cell productivity (pcd) 300 kDa glycoprotein-Fc fusion low 5 to 7 ~4 bispecific antibody medium ~16 7 to 12 antibody medium 20 to 25 35 to 50 antibody high 35 to 45 50 to 70 Stability of Stable Pools 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. Productivity Maintained Copy Number Maintained 3500 50 45 3000 40 Graphs show the maintenance 2500 35 of productivity and transposon 2000 30 copy number in clonal cell lines 25 after 60 population doublings 1500 20 1000 15 (PD). A subset of data taken 10 from 47 clones created using 500 Copy number/reference 5 Productivity mg/L Productivity Leap-In technologies is shown. 0 0 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 All clones retained at least 70% Clones Clones of their initial productivity and T0 PD60-Gln PD60+Gln T0 PD60-Gln PD60+Gln transposon copy number. Productivity and copy number is maintained after 60 population doublings. 4 201903 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 Marker promoter transposase for good levels of expression. The Mammalian marker Promoter 1 pD3600s are compatible with both of our ORF1 signal Leap-In transposases (LPN-1 and LPN-2), and Marker polyA we recommend that they only be used with ORF1 a transposase. This is because we have ORF1 poly A deliberately attenuated the selectable Insulator intervening sequence markers in the pD3600s, so multiple integration events are required for cell Leap-In Transposon viability under selection. The pD3600s P kanamycin are therefore capable of higher expression Promoter 2 yields. M_Kanamycin-r_* The pD2500 and pD3600 vectors are constructed in a modular way that allows ORF2 signal easy modification of component elements. Bacterial Origin ORF2 Versions are available that allow expression Transposon right border ORF2 polyA Insulator 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 markers ⬤ RNA processing elements Glutamine synthase (GS), dihydrofolate Introns, post-transcriptional response reductase (DHFR) elements ⬤ Drug-resistance markers ⬤ Insulators puromycin, blasticidin, hygromycin, neomycin, Flanking the construct, and isolating zeocin transcriptional units from each other ⬤ Promoters EF1a, CMV (murine and human), hybrids 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.
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