Highly Efficient Editing with CRISPR-Cpf1 in Primary T Cells and Hscs

Highly efficient editing with CRISPR-Cpf1 in primary T cells and HSCs

John Zuris, Ramya Viswanathan, Katherine Loveluck, Elise Keston-Smith, Justin Fang, Aditi Chalishazar, Jack Heath, Jen DaSilva, Eric Tillotson, Derek Cerchione, Barrett Steinberg, Fred Harbinski, Grant Welstead, Jennifer Gori, Hari Jayaram, Vic Myer

Abstract AsCpf1 selected from several tested Cpf1 orthologs AsCpf1 screen in primary T cells yields several hits a) b) Screening Target #2 with WT AsCpf1 and engineered PAM variants Cpf1 orthologs in U2OS cells Cpf1 orthologs in primary T cells The CRISPR-Cpf1 (Cas12a) system (1) offers 100 100 100 several potential advantages over other 80 nucleases for ex vivo genome editing 80 90 60 60 therapies, including a smaller single crRNA 80 that can be readily synthesized, the ability to 40 40 70

target T- and C-rich PAMs with the wild-type 20 20

% Editing by NGS by Editing % % Editing by NGS by Editing % protein and engineered PAM variants (2), and 0 0 60 MS 5 MS 11 MS 18 MS 5 MS 11 MS 18 a 5’-staggered cut which may lead to different AsCpf1 LbCpf1 Lb2Cpf1 AsCpf1 LbCpf1 Lb2Cpf1 ~ 30% hit rate repair outcomes. FnCpf1 MbCpf1 SpCas9 FnCpf1 MbCpf1 SpCas9 50 c) d) cytometry flow Selection of crRNA DR for AsCpf1 Optimization of AsCpf1 protospacer 40 For ex vivo delivery, the use of 100 100

ribonucleoprotein (RNP) complexes may be 80 80 by KO % 30 preferable, in many instances, to nucleic acid 60 60 based delivery such as plasmid DNA. Here we 20 40 40 show that several Cpf1 orthologs can be made 10

20 20 % Editing by NGS by Editing % as RNPs and edit robustly at multiple genomic NGS by Editing %

loci that are also targetable by SpCas9 (3) in 0 0 0

RR-1 RR-2 RR-3 RR-4 RR-5 RR-6 RR-7 RR-8 RR-9

WT-1 WT-2 WT-3 WT-4 WT-5 WT-6 WT-7

RR-10 RR-11 RR-12 RR-13 RR-14 RR-15 RR-16 RR-17 RR-18 RR-19 RR-20 RR-21 RR-22 RR-23 RR-24 RR-25 RR-26 RR-27 RR-28 RR-29 RR-30 RR-31 RR-32 RR-33 RR-34

RVR-2 RVR-3 RVR-4 RVR-5 multiple cell types. We demonstrate editing RVR-1

over 90% in T cells and over 90% in HSCs with Figure 1. AsCpf1 editing efficacy is superior to other orthologs and comparable to Figure 2. Screening a T cell therapeutic target with AsCpf1 and its RR and RVR PAM AsCpf1 and its engineered RR and RVR PAM SpCas9 at matched sites in a) U2OS cells and b) primary T cells. c) The AsCpf1 crRNA variants. ~30% of gRNAs show >50% editing in our preliminary screen which is on variants. direct repeat (DR) shows best efficacy in the RNP format but can be substituted with par with generally observed SpCas9 hit rate, showing that Cpf1 can potentially be ortholog crRNAs. d) AsCpf1 RNP-based cell editing is optimal with 20-nt protospacer. used for gene editing on a patient’s T cells at a key therapeutic locus. We also demonstrate optimization of the Cpf1 RNP complex, both at the protein and guide Efficient editing in HSCs and T cells by optimization of NLS configuration and nucleofection conditions level, which improve efficacy across cell a) b) c) Editing at MS 5 with NLS variants: T cells Nucleofection optimization: T cells types. Collectively, these findings underscore Pulse code #1 His-AsCpf1-nNLS 100 Pulse code #2 the promise of RNP delivery for Cpf1 80 nucleases for genome editing therapeutics. His-sNLS-sNLS-AsCpf1 Edited T cell His-sNLS-AsCpf1 60 Cpf1 variants expand targeting space for His-AsCpf1-sNLS-sNLS 40 His-AsCpf1-sNLS gene editing 20 His-AsCpf1 NGS by Editing % 0 Cpf1 Variant, PAM 0 20 40 60 80 100 Target #2 Target #3 % Editing by NGS d) Gene Editing e) f) WT, TTTV Editing at MS 5 with NLS variants: HSCs Nucleofection optimization: HSCs 50 His-AsCpf1-nNLS Pulse code #1 Pulse code #2 RR, TYCV/CCCC 40 Bone His-sNLS-sNLS-AsCpf1 + Marrow CD34 HSC RVR, TATV His-sNLS-AsCpf1 30 His-AsCpf1-sNLS-sNLS 20 His-AsCpf1-sNLS 10

Robust pipeline for RNP evaluation His-AsCpf1 NGS by Editing % Erythroid 0 Myeloid Lymphoid 0 20 40 60 80 100 WT-3 RR-4 RR-8 RR-11 RVR-1 Express CRISPR protein Purify proteins using three % Editing by NGS of interest in bacteria chromatography steps Figure 3. a) CAR and TCR engineered T cell therapies have the potential to be transformative additions to the immuno-oncology landscape. b) Optimization of NLS configuration for Cpf1 RNP delivery to primary T cells. c) Changes in the nucleofection pulse code improve maximal editing significantly in T cells at multiple therapeutic target loci d) Edited HSCs Ni-Affinity High Trap SP SRT10 300 have the potential to provide a durable therapy for patients with b-hemoglobinopathies. (e) Optimization of NLS configuration for Cpf1 RNP delivery to HSCs. f) Changes in the Column Column S.E.C nucleofection pulse code improve maximal editing significantly in HSCs when editing at a hereditary persistence of fetal-hemoglobin inducing site in HSCs.

Efficient single and multiple knockout editing in primary T cells at disease relevant loci with Cpf1 RNPs

a) b) c) d) Confirm purity by SDS-PAGE 250 RNP-enabled cell-based therapeutic Optimized NLS configuration Single KO at key T cell targets Double KO at key T cell targets 150 100 100 E R n P N d l a o T cell Target #1 A E t n " RNAf -guided n o G u r g

75 c m u l i i e # d n f o a e e r s d endonuclease# C e e R r I " S m G P

R His-AsCpf1-nNLS

R 80 u u N # i l M d A t i e e p d l i

RNAc guide e i

n 7.4 " 11.1 e c s Confirm RNP complex Benchmark in vitro Assess editing o His-AsCpf1-sNLS-sNLS m p o

R 8.3 formation by DSF specific activity efficacy in cells n i e b P o n a 60 n r t t u s

i 0.0020.0040.0060.0080.00100.00 c c " l t l e e o " o

( RNP " R p H r E N

25 WT SpCas9 o S % Editing by NGS l P t e e C c ) i n t r " " o s p e o n r

a Electroporation

15 s t i i o t

i 40 n v i

t % KO by flow by KO % y " ( P

5 m a C t

a e 73.1 i

e Patient l i l n n

RFU T cell Target #3 t t a i -5 35 40 45 50 55 60 n cell " c e

l 20

Position in AsCpf1 protospacer l

" Fraction cleaved Fraction

v His-AsCpf1-nNLS i

o His-AsCpf1-sNLS-sNLS t g " E i e i d t n i e t n a e s t " n M " e Target #1 KO Target #3 KO

C Engineered e c r RNP treatment (nM) d i e c e 0 y i n

Apo RNP d l e ) l

13 patient cell Double KO Unedited Position in SpCas9 protospacer 0 20 40 60 80 100 Target #1 Target #2 Target #3 % Editing by NGS

References: Figure 4. a) RNP workflow for an ex-vivo cellular therapy. b) NLS optimization work with MS 5 guide yields NLS variant that is shown to improve editing for multiple T cell targets. c) 1. Zetsche et al. Cell 2015 Efficient single KO at multiple therapeutically relevant T cell loci using AsCpf1 or an engineered PAM variant. d) Highly efficient double KO of two therapeutic targets in T cells 2. Gao et al. Nat Biotech 2017 treated with Cpf1 RNP as measured by flow cytometry. 3. Kleinstiver et al. Nat Biotech 2016