Deep Screening of Guide RNAs Enables Therapeutic RNA Editing with Endogenous ADAR Brian J. Booth, Lina Bagepalli, Jason Dean, Stephen Burleigh, Susan Byrne, Richard Sullivan, Yiannis Savva, Adrian W. Briggs

Summary Our Approach Results • ShapeTX developed RNAfixTM-HTS, a powerful discovery platform that enables RNAfixTM-HTS profiles RNA secondary structures to identify optimal gRNAs. RNAfixTM-HTS unlocked editing across three clinical targets. therapeutic ADAR-based RNA editing by identifying gene-encoded ADAR guide d) The ADAR-treated library TM RNAs (gRNAs) that can redirect endogenous ADAR to sites of G->A mutations. Variable gRNA A-C mismatch gRNA 112,000 gRNAs screened RNAfix -HTS gRNA TM is sequenced with NGS to • RNAfix -HTS overcomes current technological limitations by profiling up to identify promising gRNAs. hundreds of thousands of structurally unique gRNAs for each given target, to Constant target RNA LRRK2 identify designs that create ADAR-optimal substrates when bound to that target. a) For each novel target, a large G2019S • RNAfixTM-HTS applied to three targets of interest generated completely novel range of structurally randomized gRNA sequences that allow highly efficient and specific ADAR editing of each gRNAs are designed. % editing % editing target, showing the potential of a long-lasting therapeutic approach that will not require chemically modified gRNA or sequence-engineered ADAR. • LRRK2 G2019S is a G->A mutation that causes familial Parkinson’s disease. RNAfixTM-HTS identified novel gRNAs that enable ADAR-based editing of this mutation with much higher Introduction efficiency and specificity than with a conventional A-C mismatch gRNA.

2,500 gRNAs screened TM • Therapeutic RNA editing by redirecting natural ADAR enzymes b) A library of the variant gRNAs is c) The entire library is treated with A-C mismatch gRNA RNAfix -HTS gRNA has huge promise as a safe method of gene therapy without the created and bound to the target RNA. human ADAR enzyme. “5’G” that inhibits novel gRNA target editing overcomes 5’G risk of DNA damage and with no delivery of non-human proteins. ABCA4 problem e) Each library is sequenced over a time course of ADAR exposure, generating a high resolution dataset • ShapeTX’s RNAfixTM technology delivers a gRNA that hybridizes to containing kinetic editing curves for every adenosine within each unique gRNA/target complex. gRNAs G1961E

a clinically-relevant target mRNA and recruits endogenous ADAR. enabling rapid on-target editing with slow to zero off-target editing are prioritized for further validation. % editing % editing ADAR then deaminates the target adenosine to inosine, which is thousands of tested gRNAs fast read by cellular machineries as guanosine. 30 sec editing • ABCA4 G1961E is a G->A mutation that causes Stargardt disease. Unfortunately, the target This gRNA shows rapid target adenosine sits adjacent to a 5’ guanosine, which is known to strongly disfavor ADAR editing. • Whereas ADAR gRNAs can be delivered as chemically modified oligos, gene-encoded gRNAs 1 min base editing kinetics (left), and Despite this, RNAfixTM-HTS yielded designs that enable ADAR to edit this “5’G” target. high specificity for the target % editing delivered by AAV carry substantial potential advantages over oligonucleotides by providing % editing 3 min base vs off-target bases (right). non-toxic, long-lasting and even one-time treatments for serious diseases. time (min) target sequence A-C mismatch gRNA 69,000 gRNAs screened RNAfixTM-HTS gRNA 10 min • However, gene-encoded gRNAs must be at least ~60 nucleotides long to recruit endogenous This gRNA shows slow target ADAR, risking off-target editing of adjacent adenosines. In addition, endogenous ADAR has local base editing kinetics (left), and SERPINA1 30 min low specificity for the target % editing sequence and structural preferences that can inhibit editing of many on-target adenosines. % editing base vs off-target bases (right). E342K 100 min time (min) target sequence slow % editing % editing 1.6 gene A 3.4 gene B 2.4 gene C gRNAs of 3.2 editing 3.0 2.2 target adenosine increasing length 1.4 2.8 2.0 2.6 2.4 1.8 2.2 1.2 2.0 1.6 1.8 1.4 Luciferase 1.6 TM • SERPINA1 E342K is a G->A mutation that causes alpha-1 antitrypsin deficiency (AATD). An A-C 1.0 1.4 1.2

Portion of gene X Target increase fold 1.2 1.0 RNAfix -HTS can distinguish gRNA performance with ADAR1 vs ADAR2. 1.0 mismatch gRNA shows low efficiency editing, and low specificity perhaps due to the presence of

adenosine activity luciferase 0.8 0.8 0.8 20 30 40 50 60 100 150 200 20 30 40 50 60 100 150 200 20 30 40 50 60 100 150 200 • 112,000 gRNAs against LRRK2 G2019S displayed a wide range of editing patterns between the target adenosine TM A reporter assay was used to detect ADAR-based editing of three different gRNA length / nt many local adenosines. RNAfix -HTS generated gRNAs with high efficiency and specificity. target sequences cloned upstream of a luciferase open reading frame. and an off-target adenosine at position -2 (i). ADAR1 and ADAR2 showed overlapping but distinct gRNA preferences (ii), highlighting the need to profile both ADARs when targeting tissues where they are both present.

• The conventional solution for maximizing both ADAR1 ADAR2 Conclusions 40 on-target editing i) 100 100 ii) ADAR1 efficiency and specificity of endogenous ADAR at ADAR2 TM 30 off-target editing 80 80 • RNAfix -HTS enabled efficient and specific RNA editing by endogenous ADAR

a target base is to place a single A-C mismatch 60 60 # gRNAs (from 112,000) where: across several distinct sequence contexts, including challenging “5’G” adenosines. 20 Position 0 editing > 80% between the gRNA and target adenosine. 10 40 40

% editing AND Position -2 editing < 20% TM However, this approach is clearly insufficient for 0 20 20 • The datasets generated by RNAfix -HTS will be well suited for machine learning position 0 editing 0 position position 0 editing 0 position the vast majority of clinical target adenosines. The editing profile of a target region in the RAB7A gene when a 0 0 techniques to derive predictive models and further improve gRNA performance. 100-mer “A-C mismatch” ADAR gRNA is transfected into human cells. 0 20 40 60 80 100 0 20 40 60 80 100 position -2 editing position -2 editing

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