Literature Seminar #1

Intracellular delivery of CRISPRCas9 RNP for medical applications

211104B4 Yuto Azumaya

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①Introduction

1) CRISPR/Cas9 system
2) Expectations for medical applications
3) other genome editing tools
4) Delivery form of CRISPR/Cas9 system
5) Investigational drugs using CRISPR-Cas9② Methods of delivering CRISPR/Cas9 RNP
・③ Delivery of systems that cause HDR with high yields④Summary

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CRISPR/Cas9 system

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•A tool that can perform DNA editing•Consists of Cas9 protein and gRNA
•The gRNA recognizes and cleaves the targetsequence.
•Cleaved sequences are repaired mainly by end
joining (NHEJ) or homologous recombination(HDR)•NHEJ is prone to errors such as deletions andmutations → suitable for gene deletions•HDR can introduce the desired sequence
depending on the design of the donor DNA.

(Nataša Savić, Gerald Schwank,Translational Research,Volume 168,2016,15-21,)

Kim S, Broströmer E, Xing D, et al.Science. 2013;339(6121):816-819..
Ramirez-Phillips, A.C., Liu, D. AAPS J 23, 80 ,2021.

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Expectations for medical applications of the CRISPR/Cas9 system

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Diseases for which CRISPR-Cas9 system application is expected
・ Diseases caused by genetic abnormalities: Editing pathogenic genes for radical treatment
Ex)・Huntington‘s disease: nucleotide elongation disorder, CAG repeat expansion of the huntingtin gene・Hemophilia: loss of clotting factors due to mutation
・Duchenne muscular dystrophy: generation of loss-of-function proteins by frameshifting・ sickle cell disease :deformation caused by a single Glu6Val mutation in the HBB geneetc.
・Viral diseases: Disrupting viral genes to inhibit their functionEx)
・HSV・HIV・HPV・HBVetc.
・ Cancer: Treat cancer by deleting oncogenes or introducing tumor suppressor genes
Ramirez-Phillips, A.C., Liu, D. AAPS J 23, 80 ,2021.

4other genome editing tools and advantages of CRISPR/Cas9

ZFN

TALEN

Fok-1 nuclease+ Zinc finger protein(recognizes the targetsequence)

Fok-1 nuclease+TALE domain (Base isrecognized by amino acidsequence of RVD variableregion)

sequence-specific DNA-binding protein→difficult and expensive to engineer.

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CRISPR/Cas9Cas9 nuclease+ sgRNA(targeting genome)
・RNA : easily synthesizable& appliable to other genes bysimply changing the RNAsequence.

Ramirez-Phillips, A.C., Liu, D. AAPS J 23, 80 ,2021.

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Delivery form of CRISPR/Cas9 system

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Cong-Fei Xu et al.Advanced Drug Delivery Reviews, Volume 168,2021,Pages 3-29,

6Investigational drugs using CRISPR-Cas9211104 LS#1

EDIT-101
・Target disease: LCA10 (leber congenital amaurosis
type 10)
・The IVS26 mutation in the CEP290 gene causes
abnormal splicing, resulting in the addition of extra
sequences to the mRNA → loss of function.
・Deletion or reversion of the mutation by cutting
across the mutation site cures the splicing
abnormality and allows normal CEP290 to be
produced.
・Introduced by AAV in the form of pDNA・Vitro: average productive editing rate (reverse +deletion) 16.6±6.5%
・Vivo: up to 28% in subretinal injection incynomolgus monkeys (10% deletion thought to leadto visual recovery)
・Clinical trial (ongoing Phase 1, initial results):
confirmed efficacy signal in 2 out of 3 patients inmedium volume cohort

Maeder, M.L.,et al. Nat Med 25, 229–233 (2019).
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Investigational drugs using CRISPR-Cas9211104 LS#1

• NTLA-2001• Target disease: ATTR amyloidosis• accumulation of misfolded transthyretin (TTR) protein →nerve damage orheart muscle disease• TTR KO → decrease in the production of TTR• lipid nanoparticle encapsulating Cas9 mRNA + sgRNA• Introduced by intravenous infusion• Vitro (primary human hepatocytes): Editing efficiency of more than 93.7%,
decrease in the production of TTR of more than 95%, no off-target editing
confirmed• Vivo (cynomolgus monkeys): up to 73% editing efficiency, >94% serum
TTR reduction, no off-target editing• Clinical trial (on-going Phase 1, interim data): Sustained reduction in serum
TTR protein concentration (up to 87%) was observed.
Gillmore, J. D., Gane, et al.New England Journal of Medicine, 385(6), 493–502.

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Delivery form of CRISPR/Cas9 complex

・stable・simple to produce
・easy to deliver(ex vivo with physicalmethods)(in vivo with viral vectors)
・Long-term Cas9 expression
by transcription and translation→off-target DNA cleavage /host immune responses
・Possibility of insertional
mutation

211104 LS#1・limitation of the durationof Cas9 activity→Reduction of off-targets
・Does not cause
insertional mutation
・Low stability of mRNA・Requiring Translation

・No transcription and translation→Editing is possible even for cells with lowtranscriptional and translational activity.
・Easy to control the amount of transfection
・Quick editing
・High activity can be expected. (binding tosgRNA is not inhibited)
・Lowest possibility of off-target cleavage
・Reduction of immune response
・No insertional mutation

Cong-Fei Xu et al.Advanced Drug Delivery Reviews, Volume 168,2021,Pages 3-29,

9How to deliver?

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Zhang S, Shen J, Li D, Cheng Y. Theranostics 2021; 11(2):614-648

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①Introduction② Methods of delivering CRISPR/Cas9 RNP

1) General
2) Cationic substances commonly used for membrane permeation
3) Synthetic lipid nanoparticles
4) polymers
5) Inorganic nanoparticles
6) CPP conjugation
7) Biological Production③ Delivery of systems that cause HDR with high yields④Summary11

Various methods of delivering CRISPR/Cas9 RNP

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↓ Form nanoparticles↓ Coating or attaching with membrane
permeable molecules

Basic design concepts

or・Membrane permeability: cationiclipids, PEI, CPP, etc.
・Nanoparticle formation: polymer,metal, DNA, etc.
Cong-Fei Xu et al.Advanced Drug Delivery Reviews, Volume 168,2021,Pages 3-29,

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Cationic substances commonly used for membrane permeation
・ Cationic lipids+
cationic headgroup ++linker

hydrophobic portion+ +・PEI
Polyethyleneimine, a polymer

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In both cases, uptake occurs by the following
mechanisms
・Interaction with membranes→Endocytosis→Escape from endosomes※All of them are cytotoxic.

・CPP
Peptides with cell membrane permeability
Many of them contain a large number ofcationic residues.

Ruseska, I.; Zimmer, A. Beilstein J. Nanotechnol. 2020, 11, 101–123. 13

Methods of delivering CRISPR/Cas9 RNP ①: Synthetic lipid nanoparticles

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・the Cas9/sgRNA RNP is negatively charged→Encapsulated by lipid molecules in liposomes.
・Most commonly used in research applications.
Ex) lipofectamine CRISPRMAX(Commercially available reagents dedicatedto CRISPR transfection, the genome
editing efficiencies achieved 55%, 75%
and 85% in human iPSCs, mouse EScells and HEK293FT cells)
・HDR achieved

Zuris, J., Thompson, D., Shu, Y. et al. Nat Biotechnol 33, 73–80 (2015).

14Methods of delivering CRISPR/Cas9 RNP ② : polymers

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• Form complexes with Cas9 RNPs using polymers
Advantages
・Easy to synthesize
・Flexible structure and composition
・Easy to functionalize
・DegradableEx) PBA-rich dendrimer

Interaction of phenylboronic acid (PBA) withproteins to form a more stable complex
・20~40% in del (293T-EGFP cells). 15

Zhang S, Shen J, Li D, Cheng Y. Theranostics 2021; 11(2):614-648

Methods of delivering CRISPR/Cas9 RNP ② : polymers
PEGylated PLL

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PBAE(poly(β-amino ester) nanoparticles)

・HDR achieved

Chitosan (CS) nanoparticles

・HDR achieved. 16

Zhang S, Shen J, Li D, Cheng Y. Theranostics 2021; 11(2):614-648

Methods of delivering CRISPR/Cas9 RNP ③ :nanogel

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nano capsule

G. Chen, A.A. Abdeen, et al. Nat. Nanotechnol., 14 (2019), pp. 974-980

・18.7% in del. 17

Zhang S, Shen J, Li D, Cheng Y. Theranostics 2021; 11(2):614-648

Methods of delivering CRISPR/Cas9 RNP ④ : Inorganic nanoparticles

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• Use inorganic particles, such as metals, to form the complex.
AuNP

・HDR achieved(~5%)

Lee, K., Conboy, M., Park, H.M. et al.Nat Biomed Eng 1, 889–901 (2017)18

Methods of delivering CRISPR/Cas9 RNP ④ : Inorganic nanoparticles
MOF(Metal-organic frameworks)

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BP(Black Phosphorus Nanosheets)

・30~60% in del・HDR achieved

Shahad K. et al.
Journal of the American Chemical Society 2018 140 (1), 143-146

・17~32% in delW. Zhou, H. Cui, L. Ying, X.-F. Yu, Angew. Chem. Int. Ed. 2018, 57, 10268.

GO(graphene oxide)・30~60% in delYue, H., Zhou, X., Cheng, M., & Xing, D. (2018). Nanoscale, 10(3), 1063–1071.

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Zhang S, Shen J, Li D, Cheng Y. Theranostics 2021; 11(2):614-648

Methods of delivering CRISPR/Cas9 RNP ⑤ : CPP conjugation

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ex：Cas9 +CPP / CPP・sgRNA complex
Cas9-Smal-CPP+sgRNA-CPP complex
16% in del (HEK293T in vitro)
Ramakrishna, S., Kwaku Dad, A. B., et al.Genome Research, 24(6), 1020–1027.

ex： Cas9-LMWP-sgRNA ternary Cas9 RNP

Cas9-NLS-LMWP(CPP) protein+sgRNA

43.9% in del (A549 cells in vitro)
Seung Min Kim, et al. ACS Nano 2018 12 (8), 7750-7760

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Methods of delivering CRISPR/Cas9 RNP ⑥ : Biological Production

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• Viral vectors, extracellular vesicles, etc.
Ex)NanoMEDIC
・Expression of three viral proteins (VSV,HIV-Gag, and LM) to form virus-likevesicles
・Cas9 protein is incorporated into EVs
through interaction between FRB andFKBP・sgRNA is incorporated into EVs byinteraction between GAG and ψ⁺.
・Introduced into target cells by viralproteins on budding extracellular vesicles
・up to 92% exon skipping(DMD patientiPSCs)
Gee, P., Lung, M.S.Y., Okuzaki, Y. et al. Nat Commun 11, 1334 (2020).

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Methods of delivering CRISPR/Cas9 RNP ⑦ : DNA-211104 LS#1

・DNA nanoclew

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Short summary

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• Various delivery methods for CRISPR-Cas9 RNPs have been developed.
• Many of them form nanoparticles or covalent complexes that are permeable to cell membranes andare transferred into cells via endocytosis.
• Nanoparticle materials are diverse( Lipids, polymers, inorganic particles, peptides, etc.)

23Difficulty in causing HDR

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Most of the existing systems have focused on gene deletion by NHEJ, and few have achieved genetransfer by HDR.
・HDR achieved
・liposome：5~85% in del/ 8~16% HDR
・AuNP ：14.6~34% in del / ~5% HDR
・ MOF ：30~60% in del / 20~% HDR
・Chitosan (CS) nanoparticles ：16.9~55.8% in del /12.5 % HDR
・PBAE(poly(β-amino ester) nanoparticles) ： 47~77 % in del / 4% HDR

HDR is difficult to achieve with existing systems
・Low efficiency compared to NHEJ → Cytotoxicity of carriers (especially cationic career) occurs
when trying to increase the concentration
・Co-localization of donor DNA is required(NHEJ：only RNP is required).

Zhang S, Shen J, Li D, Cheng Y. Theranostics 2021; 11(2):614-648

Yoo Kyung Kang, et al.
Journal of Industrial and Engineering Chemistry,Volume 102,2021,Pages 241-250,

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Challenge to create a system that can generate HDR with high yield211104 LS#1

• As few carriers as possible → Aim to reduce toxicity• Interaction and complexation with ssODN by charge→Enables co-localization with donor DNA
Yoo Kyung Kang, et al.
Journal of Industrial and Engineering Chemistry,Volume 102,2021,Pages 241-250,25

Challenge to create a system that can generate HDR with high yield

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・Low cytotoxicity→Higher concentration could be achieved
・HDR : up to 31% (exceeding CRISPRMAX)
・Still limited to use in vitro → awaiting vivo application
20nM

200nM

Yoo Kyung Kang, et al.
Journal of Industrial and Engineering Chemistry,Volume 102,2021,Pages 241-250,26

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①Introduction② Methods of delivering CRISPR/Cas9 RNP③ Delivery of systems that cause HDR with high yields④Summary27

Summary

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introduction• CRISPR-Cas9 is attracting attention as a powerful tool for genome editing therapy.
• Drug discovery using Cas9 has already begun, and some have advanced to clinical trials.
• Delivery of Cas9 in the form of RNPs has many advantages over other methods, and delivery
methods are being developed to achieve these advantages.

Methods of delivering CRISPR/Cas9 RNP• Intracellular delivery of CRISPR-Cas9 RNPs is generally performed by linking them with membranepermeable materials.
• The materials of the complexes can be lipids, polymers, inorganic particles, peptides, etc.
• Most of the currently developed delivery methods target only deletion editing, and only a limited
number of systems are capable of HDR-mediated gene transfer, but the development of delivery
methods that enable this is underway.

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