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Transthyretin amyloidosis (ATTR) is a progressive disease caused by accumulation of amyloid deposits of misfolded transthyretin (TTR) protein in multiple tissues including the heart, nerves and gastrointestinal tract. Reduction of TTR monomer via stabilization of circulating tetramer and silencing of TTR gene expression in hepatocytes of ATTR patients have emerged as successful therapeutic strategies for chronically-administered medicines. As such, specific disruption (or knockout) of the TTR gene in hepatocytes using the CRISPR / Cas9 gene editing system is a potentially attractive next-generation treatment for ATTR, which may durably reduce the expression of TTR without the need for chronic therapy.

Liver editing was determined by NGS from a core needle liver biopsy and circulating serum TTR concentration was determined by an LC-MS / MS assay specific for the TTR protein.

Ionizable lipid concentration was determined by an LC-MS / MS assay. gRNA and Cas9 mRNA were measured by a qRT-PCR assay with primers and probes specific for the analyte.

Development of NTLA-2001, a CRISPR / Cas9 Genome Editing Therapeutic for the Treatment of ATTRKristy Wood1, Melissa Pink1, Jessica Seitzer1, Noah Gardner1, Seth Alexander1, Tracy DiMezzo1, Adam Amaral1, Samantha Soukamneuth1, Arti Kanjolia1, Rubina Parmar1, Ellen Rodhe1, Reynald Lescarbeau1, Cindy Shaw1, Tanner Dirstine1, Carri Boiselle1, Kathryn Walsh1, Bo Han1, Maria Saraiva2,3, Eva Essig1, John Leonard1, Michael McCaman1, Yong Chang1

1Intellia Therapeutics, Cambridge, MA, USA; 2Instituto de Investigacao e Inovacao em Saude, Universidade do Porto, Porto, Portugal; 3IBMC – Institute for Molecular and Cell Biology, Porto, Portugal

INTRODUCTION RESULTS

RESULTS

METHODS

• Large cargo capacity for CRISPR / Cas9 • Transient expression • Scalable synthetic manufacturing • Redosing capability • Low immunogenicity, well-tolerated, & biodegradable

• >97% serum TTR reduction is maintained at 12 months following a single administration as measured by ELISA specific for TTR protein

• No transformation or neoplastic changes observed across >100 mice over time up to 12 months post-editing

• Semi-quantification of human TTR by immuno-histochemistry in stomach, colon, sciatic nerve and DRG’s.

• Approximately 85% or better reduction in TTR staining was observed across these tissues.

Mouse model from Santos et al. 2003; In collaboration with U. of Porto

• A single administration of TTR LNP resulted in >99% reduction of TTR in plasma at 8 weeks

• Changes in peripheral TTR levels (plasma) had no effect on TTR levels in the CSF of treated mice

Mouse model from Santos et al. 2003; In collaboration with U. of Porto

CONCLUSIONS• NTLA-2001 is advancing toward the clinic in

collaboration with Regneron Pharmaceuticals, Inc with an IND submission planned for mid-2020

• TTR LNPs enable significant knockdown of the TTR protein by editing of the TTR gene across multiple species, including mouse and NHP

• In NHP, achieved a therapeutically meaningful level of TTR protein reduction that correlated with robust and significant editing in the liver

• Cas9 mRNA, sgRNA and ionizable lipid are quickly cleared from circulation, with the lipid having plasma and liver half-lives of 20 hours and 17 hours, respectively, in NHP

• Following a single dose of LNP-delivered CRISPR /Cas9 in mice:

– Editing levels achieved that resulted in >97% reduction in circulating serum TTR protein

– Reduction of circulating levels of TTR sustained for at least 12 months

– No significant histopathology findings noted• Humanized mouse model of hATTR that expresses

the V30M mutant form of the human TTR protein demonstrated rescue of TTR deposition in multiple tissues after a single dose of LNPs containing the CRISPR / Cas9 components

• Demonstrated the potential of LNP delivered in vivo CRISPR / Cas9 gene editing; suggests that future therapies based on this platform may enable next-generation, curative treatment paradigms for chronic genetic diseases such as ATTR

Lipid Nanoparticles (LNPs)

Key Advantages of LNP Delivery

Objective: To develop NTLA-2001, a lipid nanoparticle (LNP) formulated CRISPR / Cas9 genome editing therapeutic targeting the human TTR gene for the treatment for ATTR. NTLA-2001 is advancing toward the clinic with an IND submission planned for mid-2020.

Achieved Therapeutically Relevant and Sustained Serum TTR Protein Reduction in Non-Human Primate (NHP) After a Single Dose of TTR LNPs

TTR LNPs and Cargo Exhibit 17 – 24 Hour T1 / 2 and Are Cleared from Circulation and Liver Within 5 days in NHP

Findings from huTTR V30M Mouse Model Study Recapitulate TTR Deposition Phenotype in Tissues and the Nervous System

Decreasing Serum TTR by Editing the huTTR V30M Mouse Model Via TTR LNP Dramatically Decreases TTR Deposition in Tissues

Achieved Persistent Serum TTR Protein Reduction for 12 Months in Mouse After a Single Administration of TTR LNPs with No Histological Findings

Liver Editing by Next Generation Sequencing (NGS): Genomic DNA (gDNA) was isolated from livers by homogenizing a liver biopsy. gDNA samples are sequenced (NGS) using amplicon sequencing directed to the site of interest. The editing percentage is defined as the total number of sequence reads with indels or substitutions divided by the total number of sequence reads, including wild-type.

CRISPR / Cas9

Modular Platform

LNP

RNA

Genetic diseases

In VivoCRISPR / Cas9 Delivery

CRISPR is the therapyCRISPR / Cas9

KNOCKOUTKnockout of toxic or compensatory genes

(i.e. TTR)

Cas9 mRNA

ATTR gRNA

Lead Human TTR LNPs Demonstrate On-Target Editing, Reduction of TTR mRNA and TTR Protein in Primary Human Hepatocytes In Vitro

0.001

9531

25

0.003

9062

5

0.007

8125

0.015

625

0.031

25

0.062

50.1

25 0.25 0.5 1 2 4 8

0

50

100

150

0

50

100

150

Lead Candidate 1

Guide concentration (nM)

Editi

ng(%

)

TTRR

elativeExpression

TTRm

RN

A/G

eoMean

[GA

PDH

/PPIB]

TTRProtein

/Parental

Editing mRNA Protein

0.001

9531

25

0.003

9062

5

0.007

8125

0.015

625

0.031

25

0.062

50.1

25 0.25 0.5 1 2 4 8

0

50

100

150

0

50

100

150

Lead Candidate 2

Guide concentration (nM)

Editi

ng(%

)

TTRR

elativeExpression

TTRm

RN

A/G

eoMean

[GA

PDH

/PPIB]

TTRProtein

/Parental

Editing mRNA Protein

0 5 10 15 20 25 30 35 40 45 50 550

10

20

30

40

50

60

70

80

Weeks Post Dose

%Ed

iting

High DoseMedium DoseLow Dose

0 5 10 15 20 25 30 35 40 45 50 550

10

20

30

40

50

60

70

80

Weeks Post Dose

%Ed

iting

High DoseMedium DoseLow Dose

0 5 10 15 20 25 30 35 40 45 50 550

10

20

30

40

50

60

70

80

Weeks Post Dose

%Ed

iting

High DoseMedium DoseLow Dose

0 5 10 15 20 25 30 35 40 45 50 550

10

20

30

40

50

60

70

80

Weeks Post Dose

%Ed

iting

High DoseMedium DoseLow Dose

Homozygous for the human mutant V30M TTR transgene in a mouse TTR-null background

transgenic mice contain approximately ~47 copies of huTTR V30M

1 32muTTR muTTR huTTR V30M

huTTR V30M

0 40 80 120 1600

20

40

60

80

100

120

Time (d)

TTR

Prot

ein

(%of

base

line)

Dec 2018-Dose Level #1Control Dec 2018-Dose Level #2

Single-Dose TTR Editing

TTR EditingLiver Editing

TTR Protein ReductionSerum TTR

Ionizable Lipid

Chart includes single administration within a range of dose levels

Single dose Single dose

Therapeutically Relevant Range:>60% TTR Knockdown

TTR Protein Reduction

gRNA & Cas9 mRNA

Single Dose IVHalf Life t1 / 2 (h)

Lipid gRNA mRNAPlasma 20 21 18

Liver 17 19 24

Control HuTTR0

100

200

300

400

500

Plasma (8weeks)

TTR

(μg/

mL)

Control HuTTR0

20

40

60

Editing at the TTR locus

%Ed

iting

49.4%

Control HuTTR0

5

10

15

CSF

TTR

(μg/

mL)

not significant

Stomach

Con

trol

TTR

LNP

Colon Sciatic Nerve DRG

Vehicle High Dose

Day

712

Mos

.

Exon 2Edit / KO

mRNA

TTR protein

0 24 48 72 96 120 144 168100

101

102

103

104

105

time (h)

conc

entra

tion

(ng/

ml,

ng/g

)

gRNA plasma mRNA plasma gRNA liver mRNA liver

>95% Reduction in Circulating Levels of TTR

0 5 10 15 20 25 30 35 40 45 50 550

20

40

60

80

100

120

140

Weeks Post Dose

%of

pre-

dose

TTR

High DoseMedium DoseLow Dose