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Therapeutic Levels of FVIII Generated by CRISPR/Cas9-Mediated In Vivo Genome Editing in Hemophilia A Mice
Alan Brooks, Karen Vo, Dariusz Wodziak, Rangoli Aeran, Keith Abe, Cornell Mallari, Valerie Guerrero, Christopher Cheng#, Andrew ScharenbergCasebia Therapeutics, San Francisco, CA and Cambridge, MA #
ASGCT 22nd Annual Meeting, 2019
Session: “Gene Therapy for Metabolic Disorders: Proof of Concept and Beyond”
April 30th, 3:30-5:15, Heights Courtyard 1Abstract #428
Disclosures
| Confidential 2
All authors are employees of Casebia Therapeutics LLC
Hemophilia A - Background
|Confidential 3
• Genetic disorder caused by non-functional FVIII gene resulting in deficiency of active FVIII protein
• Protein replacement is the SOC:• 1 to 3 x weekly i.v. infusions• Peaks and troughs of FVIII resulting in less than ideal protection
• Ideal therapy is a constant level of FVIII in the normal range (50-150%) to provide continuous hemostasis
• AAV based gene therapy shows promise in clinical trials but may have limitations:• Life-long persistence of episomal based vectors un-proven• Only 1 treatment possible (due to nAB to AAV) with variable and unpredictable FVIII levels• Concern about utility in pediatrics due to vector dilution
• Approach presented: • Expression from FVIII gene integrated at a defined site in the genome• Potential for titration to the normal range which may represent the ideal curative therapy• Absence of liver specific promoter/enhancer minimizes risks of insertional gene activation
Targeted Gene Insertion Mediated by CRISPR-Cas9
| Confidential
CAS9
Exon 2Exon 1Albumin
Promoter
Exon 2Exon 1 GOI (secretable protein, no SP)SA pA
AAV packaged donor
GOI (secretable protein, no SP)SA pA
Exon 2Exon 1
GO secretableprotein, no SP) SApA or
orExon 2Exon 1
NHEJ
Forward Integration:
Reverse Integration:
INDELS (no impact on Alb expression):
Hybrid mRNA
SecretedProtein
In Vivo LNP Delivery of spCas9 mRNA /gRNA Targeting Albumin
Confidential 5
• Cationic lipid, neutral lipids, PEG-lipid• Single gRNA (chemically modified)• spCas9 mRNA (with NLS)
Self assembly(PrecisionNanoSystemsflow cell)
LNP• 50 – 90 nm• RNA concentration determined by Ribogreen• Dose mice by total RNA concentration (sgRNA + mRNA)
sgRNA 1 targets intron 1 of mouse albumin
Dynamic Light Scattering
I.V. LNPOn-target INDELS at day 7
HemAMice
# Genomic DNA from liver or spleen analyzed by TIDE
#
Visit Poster Abstract #494 for more details
0 . 5 1 2 P B S0
5
1 0
1 5
2 0
2 5
D o s e ( m g / k g )
To
tal
IND
EL
%
L iv e r l e f t l o b e
L i v e r r i g h t l o b e
S p le e n
Repeat LNP Dosing
Confidential 6
Murine SEAP pASAAAV8
2e12 vg/kg injected in HemA mice
Repeated LNP dosing, gRNA 1 (2mg/kg) T1= 0 days T2= 28 days T3= 49 days
SEAP activity in plasma measured weekly after each LNP dose Mean SEAP from 3 to 4 weekly assays plotted
Stuffer
0
2000
4000
6000
8000
10000
12000
14000
16000
1 2 3
SEAP
act
ivity
(mic
roU
/ml)
T1 T2 T3# Background SEAP activity in mice that received AAV only was 0
#
• Sequential increases in gene expression after each LNP dose
• This modality has the potential to tune the desired expression level after a single dose of AAV
4.7 Kb
Therapeutic Levels of FVIII After Genome Editing in HemA mice
Confidential 7
Human FVIII cDNA pASAAAV8
AAV-FVIII + LNP (2 mg/kg) –sgRNA1AAV-FVIII only
HemAMice
• Therapeutic FVIII activity achieved, approaching the normal range
• No FVIII when only the AAV-FVIII donor was injected demonstrating that cleavage of the genome required
• Antibodies against the foreign human FVIII protein develop in some mice
AAV + LNPAAV only
1 2 3 4
0
2 0
4 0
6 0
8 0
1 0 0
FV
III
Ac
tiv
ity
(%
of
no
rma
l)
2 - 1
2 - 2
2 - 3
2 - 4
2 - 5
W e e k s p o s t L N P
*
* Antibodies to human FVIII present
Targeted Integration Correlates to Cellular Distribution of Hybrid mRNA
Confidential 8
Day 10: FVIII activity in bloodDay 25: BaseScope™ and Targeted Integration by DD-PCR
BaseScope™ for hybrid mRNA in liver
Naï
ve m
ice
AAV8
-FVI
II
+LN
P
2% to 4% ofcells positive
Z ZAlb Exon1 FVIII
Human FVIII cDNA pASAAAV8
Group(n=5)
FVIII activity on day 10(% of normal)
Targeted Integration(% per haploid genome)
AAV8-FVIII +LNP 26 +/- 9 2.5 +/- 0.6
AAV8-HLP-FVIII #
28 +/- 5(peak on d30)
-
Naïve mice 0 0
# HLP promoter driving same FVIII CDS (but with signal peptide)
AAV8-FVIII (2e12 vg/kg) + LNP (2mg/kg)AAV8-HLP-FVIII (2e12 vg/kg)
HemAMice
Human FVIII cDNA pASAIntron 1For
RevP∗Intron 1DD-PCR:
Human FVIII cDNA (with SP)HLP pAAAV8
Long Term Expression of FVIII
Confidential
9
• Immune deficient NSG mice to prevent antibodies to human FVIII• FVIII detected with human FVIII specific capture-activity assay
Weeks post LNP
FVIII
act
ivity
(% o
f nor
mal
)
2e12 vg/kg AAV8 donor + 2mg/kg LNP2e13 vg/kg AAV8 donor + 2mg/kg LNPNaïve Mice
Stable expression to 7 months and continuing to monitor
FVIII levels dependent on dose of AAV donor
AAV and LNP Dose Response in HemA Mice
Confidential 10
Human FVIII cDNA pASAAAV8 +/- LNP
• FVIII level can be modulated by LNP dose
• Higher doses of AAV enable FVIII expression at lower LNP doses
• FVIII levels correlate to targeted integration frequency
% Mouse had antibodies to human FVIII on d14# Mouse had no INDELS indicative of failed LNP delivery
G 1 G 2 G 3 G 4 G 8 G 9 G 1 0
0
2 0
4 0
6 0
FV
III
Ac
tiv
ity
on
da
y 1
4
(%
of
no
rma
l)
A A V D o s e ( v g / k g ) : 2 e 1 2 2 e 1 2 2 e 1 2 2 e 1 2 1 e 1 2 5 e 1 2 2 . 5 e 1 3
L N P D o s e ( m g / k g ) : 0 . 5 1 2 0 1 1 1
2e12 vg/kg AAV
LNP LNP 1 mg/kg
AAV
0.010.11
0.62
0.01 0.080.19
0.53
# %
No
LNP
cont
rol
(Mean targeted integration %)
(0.01)(0.11)
(0.62)
(0.01) (0.08)(0.19)
(0.53)
2e12AAV Dose (vg/kg): 2e12 2e12 2e12 1e12 5e12 2.5e130.5 1 2 0 1 1 1LNP Dose (mg/kg):
Selection of gRNA can Impact Expression
Confidential 11
Human FVIII cDNA pASAAAV8
Optimized LNP, two gRNA targeting Albumin intron 1 gRNA1 (used for results in previous slides) gRNA2
FVIII Day 8 post LNP
• 5-fold improved expression using gRNA2 vs gRNA1• Improvement with gRNA 2 was not due to higher INDELS• 100% FVIII with low variability achieved with 2e12 vg/kg AAV8 donor• Measurement of targeted integration is planned
LNP only (1.5 mg/kg)AAV-FVIII (2e12 vg/kg) + LNP (1.5 mg/kg)
HemAMice
Mice dosed with LNP only
g R N A 1 g R N A 20
2 0
4 0
6 0
8 0
1 0 0
IND
EL
% (
d7
)
g R N A 1 g R N A 20
2 0
4 0
6 0
8 0
1 0 0
1 2 0
FV
III
Ac
tiv
ity
(%
of
no
rma
l)
Summary and Conclusions
Confidential 12
• CRISPR-Cas9 mediated targeted integration of FVIII at the albumin locus generates therapeutic levels of FVIII in mice, up to 100% of normal
• Low levels of targeted integration are sufficient for curative levels of FVIII
• Expression was stable (7 months and continuing)
• Targeted integration correlated to FVIII levels and was dependent on dose of both AAV and LNP
• FVIII level could be modulated by LNP dose
• Transient delivery of the nuclease as a LNP packaged mRNA has the potential for repeat dosing to “dial-in” FVIII levels while minimizing off-target risk
• Further optimization and evaluation in higher species in progress
Acknowledgments
Confidential 13
Chandra Patel
Luis Gamboa
Greg Cost
Gene Uenishi
Christopher Cheng
Faye Wu
Samantha Chin
Scott Munzer
Karolina Kosakowska
Kui Wang
Patrick Au
Mihail Rokas
Jim Burns
Co-Authors Additional ContributorsKaren Vo
Dariusz Wodziak
Rangoli Aeran
Keith Abe
Cornell Mallari
Valerie Guerrero
Christopher Cheng
Andrew Scharenberg