in vivo bone marrow editing...edited hscs retain their capacity to reconstitute primary...
TRANSCRIPT
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
3 | CONFIDENTIAL & PROPRIETARY
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
4 | CONFIDENTIAL & PROPRIETARY
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
5 | CONFIDENTIAL & PROPRIETARY
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
6 | CONFIDENTIAL & PROPRIETARY
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
7 | CONFIDENTIAL & PROPRIETARY
• 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
8 | CONFIDENTIAL & PROPRIETARY
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
9 | CONFIDENTIAL & PROPRIETARY
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*
Note: Guide “B” used in this experiment
10 | CONFIDENTIAL & PROPRIETARY
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
Note: Guide “B” used in this experiment
11 | CONFIDENTIAL & PROPRIETARY
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
12 | CONFIDENTIAL & PROPRIETARY
Key
Takeaways
12 | CONFIDENTIAL & PROPRIETARY
• 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