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Engineering a Self-inactivating Adeno-associated Virus ... · Vector for ARCUS Nuclease Delivery...
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A) ARCUS Nuclease Expression The amounts of nuclease expressed in mouse liver were determined for protein levels by Western Blot and mRNA levels by qRT-PCR. • Western blot results show that PEST tag insertion reduced nuclease expression to undetectable levels. TS insertion led to progressive reduction of nuclease over time (~50% for 2TS1 and ~80% for 3TS). • qRT-PCR results show that PEST tag or TS insertion reduced nuclease expression on mRNA levels. B) Nuclease Cleavage Activity The nuclease activity in mouse liver was determined by measuring the insertion and deletion (Indel) rates generated by the nuclease at the target site on the mouse genome by Amplicon-Seq and ddPCR. • The lower nuclease levels (<50%) resulted from TS and/or PEST tag insertion in AAV genome are able to generate indel levels up to 90% of NoTS. • TS and PEST tag insertion effects to reduce indel formation are additive. C) Off-targeting The Indel rates of ARCUS nuclease at three different off-target sites in mouse genome were determined by Amplicon-Seq. The lower Indel rates at off- targets suggest higher safety. Significant advances in engineering sequence specific nucleases have enabled a broad range of biomedical applications, particularly when combined with AAV, a versatile viral vector for in vivo post-mitotic cell gene delivery. The long-term expression of nuclease mediated by AAV delivery, however, raises concerns about safety and immunogenicity of nuclease-based applications. Persistent expression of the nuclease can increase the likelihood of off-target cleavage which can induce genotoxicity. Additionally, expression of an exogenous nuclease has the potential to elicit an immune response against transduced cells. Thus, it would be advantageous to limit the duration of nuclease expression following delivery. Self-inactivating AAV Our self-inactivating AAV system consists of a tissue-specific promoter, a nuclease open reading frame, and adjacent sites targeted by the nuclease. We investigated if target site (TS) and/or PEST insertion in AAV genome would reduce nuclease expression and off-targeting in vitro and in vivo with the goal of increasing safety of nuclease-based gene therapy. TITR = inverted terminal repeat; Prom = promotor; NLS = nuclear localization sequence In Vitro Experiment Using HepG2 Cells • The nuclease expression levels are determined by measuring luciferase activity which were co-expressed with nuclease in the cells after plasmid DNA electroporation. Incorporation of target sites (TS) in AAV genome led to progressive loss of luciferase signals over time, whose rate can be modulated by the locations and copy numbers of target sites. Acknowledgements: Thanks to Traci Reddick, Robert Brown, Megan Trum, Iika Arias, and Forrest Karriker for their support in animal studies. For more information, please contact Hui Li ([email protected]). Engineering a Self-inactivating Adeno-associated Virus (AAV) Vector for ARCUS Nuclease Delivery Hui Li, Adam Mefferd, Janel Lape, John Morris, Jeff Smith Precision BioSciences, Inc., Durham, NC 27701 American Society of Gene and Cell Therapy (ASGCT) • 12 – 15 May, 2020 INTRODUCTION • In vitro, our system can progressively reduce nuclease expression over time, and the decrease in nuclease expression can be impacted by the locations and copy numbers of the target site insertions. • In vivo, our self-inactivating AAV system enabled sufficient on-target cleavage (20-90%). • In addition, this system significantly reduced nuclease expression, AAV copy number and off-targeting by 50- 90%. • Our self-inactivating system has the potential to improve the safety of therapeutic applications using ARCUS nuclease with its ability to reduce both genotoxicity and nuclease immunogenicity through tunable control of nuclease levels over time. CONCLUSIONS OBJECTIVE IN VIVO RESULTS • Amplicon-Seq results showed that cleavage of three off-target sites decreased by 5-8 fold due to TS or PEST tag insertion. • TS and PEST tag reductions in off-targeting appear additive. D) AAV Copy Number Nuclease cleavage of the AAV genome was determined by comparing the AAV titers of two different qPCR primer pairs, a TS1 primer pair flanking the TS and an SV40 primer pair targeting SV40 poly A region in AAV genome. • ARCUS nucleases cleaved the TS inserted in the AAV genome, which led to significant reduction of AAV copy number. IN VITRO EXPERIMENT 0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 NoTS 2TS1 3TS ARCUS Expression by WB (A.U.) Western Blot 0% 10% 20% 30% 40% NoTS NoTS -PEST 2TS1 2TS1 -PEST 2TS2 -PEST 3TS 3TS -PEST PBS ARCUS mRNA level Relative to GAPDH (%) qRT-PCR 0% 10% 20% 30% 40% 50% 60% 70% 80% NoTS NoTS -PEST 2TS1 2TS1 -PEST 2TS2 -PEST 3TS 3TS -PEST PBS Indel Rate (%) ON-Target Nuclease Activity 0% 20% 40% 60% 80% 100% 120% NoTS NoTS -PEST 2TS1 2TS1 -PEST 2TS2 -PEST 3TS 3TS -PEST PBS Relative Nuclease Activity Relative Nuclease Activity (%) Target site 0% 5% 10% 15% NoTS NoTS -PEST 2TS1 2TS1 -PEST 2TS2 -PEST 3TS 3TS -PEST PBS Off-Target 3 0% 5% 10% 15% NoTS NoTS -PEST 2TS1 2TS1 -PEST 2TS2 -PEST 3TS 3TS -PEST PBS Indel Rate (%) Off-Target 1 0% 2% 4% 6% NoTS NoTS -PEST 2TS1 2TS1 -PEST 2TS2 -PEST 3TS 3TS -PEST PBS Off-Target 2 Chr BP ON-Target AAGACATTGGTGAGGAAAAATC chr2 134324016 Off-Target1 TATACATCTGTAAAGAAAAATA chr10 121990375 Off-Target2 TGGACATGATTAAGGAAACATC chr11 64902548 Off-Target3 TAGACATTGGTAAAGACAAATA chr1 193578707 IN VIVO EXPERIMENTAL DESIGN Target site; PEST = Tag for protein degradation Target site 3TS-PEST 2TS2-PEST 2TS1-PEST No-TS-PEST 3TS 2TS1 PC W2 W6 W10 NC No-TS ARCUS PC W2 W6 W10 NC PC W2 W6 W10 NC PC W2 W6 W10 NC 0 20 40 60 80 100 120 140 160 NoTS NoTS -PEST 2TS1 2TS1 -PEST 2TS2 -PEST 3TS 3TS -PEST PBS AAV Copy/diploid Genome AAV Copy Number by TS1 Primers -40% -20% 0% 20% 40% 60% 80% NoTS NoTS -PEST 2TS1 2TS1 -PEST 2TS2 -PEST 3TS 3TS -PEST PBS AAV Indel (%) Indel rate on AAV Genome 0 20 40 60 80 100 120 140 160 NoTS NoTS -PEST 2TS1 2TS1 -PEST 2TS2 -PEST 3TS 3TS -PEST PBS AAV Copy/diploid Genome AAV Copy Number by SV40 Primers 0% 10% 20% 30% 40% 50% 60% 70% 80% 0% 10% 20% 30% 40% 50% 60% 70% 80% AAV Indel Rate ON Target Indel Rate 2TS1 2TS1-PEST 2TS2-PEST 3TS 3TS-PEST Pearson r = 0.5417 P = 0.002 0.0 0.5 1.0 1.5 2.0 2.5 0 20 40 60 80 100 120 140 160 180 200 Relative Luciferase Activity Time after Electroporation (Hour) NoTS TS 2TS1 2TS2 3TS 5TS Progressive Loss of Luciferase Activity
Transcript of Engineering a Self-inactivating Adeno-associated Virus ... · Vector for ARCUS Nuclease Delivery...
A) ARCUS Nuclease ExpressionThe amounts of nuclease expressed in mouse liver were determined for protein levels by Western Blot and mRNA levels by qRT-PCR.
• Western blot results show that PEST tag insertion reduced nuclease expression to undetectable levels. TS insertion led to progressive reduction of nuclease over time (~50% for 2TS1 and ~80% for 3TS).
• qRT-PCR results show that PEST tag or TS insertion reduced nuclease expression on mRNA levels.
B) Nuclease Cleavage Activity The nuclease activity in mouse liver was determined by measuring the insertion and deletion (Indel) rates generated by the nuclease at the target site on the mouse genome by Amplicon-Seq and ddPCR.
• The lower nuclease levels (<50%) resulted from TS and/or PEST tag insertion in AAV genome are able to generate indel levels up to 90% of NoTS.• TS and PEST tag insertion effects to reduce indel formation are additive.
C) Off-targetingThe Indel rates of ARCUS nuclease at three different off-target sites in mouse genome were determined by Amplicon-Seq. The lower Indel rates at off-targets suggest higher safety.
Significant advances in engineering sequence specific nucleases have enabled a broad range of biomedical applications, particularly when combined with AAV, a versatile viral vector for in vivo post-mitotic cell gene delivery. The long-term expression of nuclease mediated by AAV delivery, however, raises concerns about safety and immunogenicity of nuclease-based applications. Persistent expression of the nuclease can increase the likelihood of off-target cleavage which can induce genotoxicity. Additionally, expression of an exogenous nuclease has the potential to elicit an immune response against transduced cells. Thus, it would be advantageous to limit the duration of nuclease expression following delivery.
Self-inactivating AAVOur self-inactivating AAV system consists of a tissue-specific promoter, a nuclease open reading frame, and adjacent sites targeted by the nuclease. We investigated if target site (TS) and/or PEST insertion in AAV genome would reduce nuclease expression and off-targeting in vitro and in vivo with the goal of increasing safety of nuclease-based gene therapy.
TITR = inverted terminal repeat; Prom = promotor; NLS = nuclear localization sequence
In Vitro Experiment Using HepG2 Cells• The nuclease expression levels are determined by measuring luciferase activity which were co-expressed with nuclease in the
cells after plasmid DNA electroporation. Incorporation of target sites (TS) in AAV genome led to progressive loss of luciferase signals over time, whose rate can be modulated by the locations and copy numbers of target sites.
Acknowledgements: Thanks to Traci Reddick, Robert Brown, Megan Trum, Iika Arias, and Forrest Karriker for their support in animal studies.For more information, please contact Hui Li ([email protected]).
Engineering a Self-inactivating Adeno-associated Virus (AAV) Vector for ARCUS Nuclease DeliveryHui Li, Adam Mefferd, Janel Lape, John Morris, Jeff SmithPrecision BioSciences, Inc., Durham, NC 27701
American Society of Gene and Cell Therapy (ASGCT) • 12 – 15 May, 2020
INTRODUCTION
• In vitro, our system can progressively reduce nuclease expression over time, and the decrease in nuclease expression can be impacted by the locations and copy numbers of the target site insertions.
• In vivo, our self-inactivating AAV system enabled sufficient on-target cleavage (20-90%).
• In addition, this system significantly reduced nuclease expression, AAV copy number and off-targeting by 50-90%.
• Our self-inactivating system has the potential to improve the safety of therapeutic applications using ARCUS nuclease with its ability to reduce both genotoxicity and nuclease immunogenicity through tunable control of nuclease levels over time.
CONCLUSIONS
OBJECTIVE
IN VIVO RESULTS• Amplicon-Seq results showed that cleavage of three off-target sites decreased by 5-8 fold due to TS or PEST tag insertion.• TS and PEST tag reductions in off-targeting appear additive.
D) AAV Copy NumberNuclease cleavage of the AAV genome was determined by comparing the AAV titers of two different qPCR primer pairs, a TS1 primer pair flanking the TS and an SV40 primer pair targeting SV40 poly A region in AAV genome.
• ARCUS nucleases cleaved the TS inserted in the AAV genome, which led to significant reduction of AAV copy number.
IN VITRO EXPERIMENT
0
1,000
2,000
3,000
4,000
5,000
6,000
7,000
NoTS 2TS1 3TS
ARCU
S Ex
pres
sion
by
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(A.U
.) Western Blot
0%
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NoTS NoTS-PEST
2TS1 2TS1-PEST
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PBS
ARCU
S m
RNA
leve
l Re
lativ
e to
GAP
DH (%
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qRT-PCR
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2TS1 2TS1-PEST
2TS2-PEST
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PBS
Inde
l Rat
e (%
)
ON-Target Nuclease Activity
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2TS1 2TS1-PEST
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PBS
Relative Nuclease Activity
Rela
tive
Nuc
leas
e Ac
tivity
(%)
Target site
0%
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10%
15%
NoTS NoTS-PEST
2TS1 2TS1-PEST
2TS2-PEST
3TS 3TS-PEST
PBS
Off-Target 3
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15%
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2TS1 2TS1-PEST
2TS2-PEST
3TS 3TS-PEST
PBS
Inde
l Rat
e (%
)
Off-Target 1
0%
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4%
6%
NoTS NoTS-PEST
2TS1 2TS1-PEST
2TS2-PEST
3TS 3TS-PEST
PBS
Off-Target 2
Chr BPON-Target AAGACATTGGTGAGGAAAAATC chr2 134324016
Off-Target1 TATACATCTGTAAAGAAAAATA chr10 121990375Off-Target2 TGGACATGATTAAGGAAACATC chr11 64902548Off-Target3 TAGACATTGGTAAAGACAAATA chr1 193578707
IN VIVO EXPERIMENTAL DESIGN
Target site; PEST = Tag for protein degradation
Target site
3TS-PEST2TS2-PEST
2TS1-PESTNo-TS-PEST 3TS
2TS1
PC W2 W6 W10NC
No-TSARCUS
PC W2 W6 W10NC PC W2 W6 W10NC PC W2 W6 W10NC
0
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2TS2-PEST
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/dip
loid
Gen
ome
AAV Copy Number by TS1 Primers
-40%
-20%
0%
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Indel rate on AAV Genome
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loid
Gen
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AAV Copy Number by SV40 Primers
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Pearson r = 0.5417P = 0.002
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Rel
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e Lu
cife
rase
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ivity
Time after Electroporation (Hour)
NoTSTS2TS12TS23TS5TS
Progressive Loss of Luciferase Activity
