In Vivo Bone Marrow Editing...Edited HSCs retain their capacity to reconstitute primary...

In Vivo Gene Editing of

Hematopoietic Stem and

Progenitor Cells

Sean Burns, M.D.

March 10, 2021

Keystone eSymposium: “Precision Engineering

of the Genome, Epigenome and Transcriptome"

2 | CONFIDENTIAL & PROPRIETARY

Sickle cell disease (SCD)

• Major global health issue impacting millions, caused by a mutation in the beta globin gene

• Characterized by severe pain and multi-organ injury with reduced life expectancy and quality of life

• Traditional treatments are limited; allogenic hematopoietic stem cell (HSC) transplantation is reserved as

a last resort for severely affected patients

→ Ex vivo gene editing of autologous HSCs recently demonstrated benefit for SCD in a clinical trial

CRISPR/Cas9

gene editing of

bone marrow

Sickled RBCs Healthy RBCs

N Engl J Med. 2021 Jan 21;384(3):252-260

N Engl J Med. 2017 Apr 20;376(16):1561-1573

Gene editing holds great promise for treating hereditary blood disorders


Ex vivo SCD gene editing still has significant limitations

Complex cell manufacturing process Conditioning regimen toxicity

• Immunosuppression for > 1 month,

predisposing to infection

• Risk of malignancy from chemotherapy

drugs, especially leukemia

• Risk of infertility

Implications

• Ex vivo gene editing will be limited to

highly selected SCD patients with

severe disease

• Treatment complexity will limit access

for patients in resource-poor settings

Mobilize and

harvest cells

Myeloablate

the patient

Gene

Editing

Cell

Expansion

Cryopreserve

Test & Release

Cell

Sorting

Infuse edited cells & support

patient until cells engraft


Desired features of in vivo approach

• Provides clinically meaningful, durable HSC editing

• Allows for multidosing to reach therapeutic target

• Preserves regenerative potential of edited cells

• Translatable to human HSC population

In vivo non-viral SCD gene editing could overcome these limitations

Infusion

of LNP

Lipid

Nanoparticle

(LNP)

Simplified process Improved safety and accessibility

• Avoids myeloablation and associated risks of

immunosuppression, malignancy, and infertility

– Approach could become mainstream therapy for SCD

• Avoids need for complex cell manufacturing or

extensive supportive care post-treatment

– Treatment simplicity could expand access to patients in

resource-poor settings


Intellia’s modular in vivo editing platform employs non-viral LNP delivery

Key Advantages of LNP Delivery

✓ Large cargo capacity

✓ Biodegradable / transient expression

✓ Low immunogenicity

✓ Redosing capability

✓ Scalable synthetic manufacturing

✓ Tunable tropism

Lipid Nanoparticles

Cas9 mRNA

Target

gRNA AAAA

gRNA target site specificity

defined by 20mer at 5’ end

Transient Cas9

expression from mRNA


Editing HSCs in vivo requires LNPs with bone marrow tropism

• LNPs designed, formulated, and tested in vivo to identify compositions with enhanced delivery

to bone marrow and HSCs

Infusion

of LNP

Bone marrow-tropic LNP

Infusion

of LNP

Liver-tropic LNP


• Dose dependent editing at 1 week post dosing in whole bone marrow and HSPC populations

• Levels predicted to have therapeutic benefit if achieved in patients with SCD

** Lin-Sca-1+c-Kit+ (LSK) cell population

Bone marrow-tropic LNP enables editing of mouse bone marrow as well as hematopoietic stem and progenitor cells (HSPCs)

* Blood. 2017;130(17):1946-1948.

0

1 0

2 0

3 0

4 0

5 0

Ed

itin

g (

%)

L o w D o s e M id D o s e H ig h D o s e

Level predicted to be

curative for SCD*

Whole bone marrow

Hematopoietic stem and

progenitor cells**

Note: Guide “A” used in this experiment


Editing of mouse bone marrow and HSCs is durable through at least one year

Editing Over One Year Period

• Editing was similar across all time points assessed, in both whole bone marrow and HSCs populations

• Results highlight the potential for a single-course, long-lasting therapy

112

24

36

54

0

5

1 0

W e e k s P o s t L N P D o s e

Ed

itin

g (

%)

Whole bone marrow

Hematopoietic stem cells*

Buffer

* Lin-Sca-1+c-Kit+CD34-Flk2- cell population

Note: Guide “B” used in this experiment


Editing of mouse bone marrow and HSCs increases with multidosing

• Non-immunogenic LNP delivery platform may enable stepwise “treat-to-target” approach

** Lin-Sca-1+c-Kit+CD34-Flk2- cell population

* Blood. 2017;130(17):1946-1948.

0

1 0

2 0

3 0

4 0

5 0

6 0E

dit

ing

(%

)

1 D o s e 2 D o s e s 3 D o s e s 4 D o s e s

Whole bone marrow

Hematopoietic stem cells**

Level predicted to be

curative for SCD*



Edited HSCs retain their capacity to reconstitute primary hematopoietic lineages

In a competitive transplant model using mice treated with a single dose of LNP, edited cells:

✓ Remained stable as a percentage of the bone marrow and HSC populations over a 14-week period

✓ Reconstituted healthy bone marrow with normal production of lymphoid and myeloid lineages

→ Nonviral approach preserves normal stem and progenitor cell function in mice

0

5

1 0

Ed

itin

g (

%)

P re P o s t

0

2 5

5 0

7 5

1 0 0

Co

mp

os

itio

n o

f

bo

ne

ma

rro

w (

%)

Normal Lineage DistributionWhole Bone Marrow Editing

Pre/Post Transplant

B cells

Myeloid cells

T cells



Nonviral CRISPR/Cas9 delivery supports editing of human HSPCs in mice

Harvest bone marrow

and recover CD34+ cells

Humanize mice with

healthy CD34+ cells

Dose LNP systemically

via tail vein injection

Week 21Day 0 Week 20

0

5

1 0

1 5

2 0

2 5

3 0

Ed

itin

g o

f C

D3

4+

ce

lls

(%

)

• Single in vivo dose of LNP achieved clinically significant level of editing in human CD34+ cells

• Results suggest cross-species relevance of LNP platform for genome engineering of HSPCs

Note: guide “C” used in this experiment


Key

Takeaways


• LNPs well suited to deliver CRISPR/Cas9 to hematopoietic cells

for durable in vivo gene editing

• Achieved clinically relevant levels of HSC editing in wild-type mice

• Multidosing of LNPs increased editing in stepwise manner,

potentially enabling “treat-to-target” therapeutic approach

• Edited cells retain regenerative potential and reconstitute primary

hematopoietic lineages

• Achieved therapeutically relevant levels of editing of human

CD34+ cells in a xenotransplant mouse model, highlighting cross-

species translation

Next step: Demonstrate relevance to future clinical application by

confirming activity in nonhuman primate model

In Vivo Bone Marrow Editing...Edited HSCs retain their capacity to reconstitute primary...

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