Advanced Genetic
Engineering of
Hematopoiesis for the
Treatment of Inherited
Diseases
Pietro Genovese, Ph.D.
Project Leader
San Raffaele Scientific Institute
Telethon Institute for Gene Therapy
Milan, Italy
Gene Editing WorkshopWashington DCApril 28th 2019
Harvest Hematopoietic
Stem Progenitor Cells
T Lymphocytes
NK Cells
Peripheral blood
Dendritic Cells
Microglia
Macrophages
Monocytes
Red Blood Cells
Granulocytes
Platelets
B Lymphocytes
Thymus
Gene
Transfer
Infuse into
conditioned recipient
Immuno-hematological diseases
Storage diseases
Therapeutic Potential of HSC Gene TherapyC
ancer, In
fectio
ns
Tissues
Therapeutic Potential of
Targeted Gene Editing in HSC Gene Therapy
• Targeted insertion in a safe locus
• Correct in situ mutations
– restores gene function and expression control
• genotoxic risk limited to off target activity
– circumscribed to small fraction of genome
• often constrained by
– unavailable or low efficiency of HDR
– need for DNA template co-delivery
Optimized HSPC Gene Editing Protocol
+ SR1, dmPGE2 and UM171
HDR within populations
• ~50-70% HDR editing in bulk CD34+ cells
• Independent from the nuclease platform
• Lower but substantial efficiency in
primitive cells (20-40%)
• Long-term multi-lineage engraftment of
edited HSPC
• Reproducible on HSPC from different
cell sources
IL2RG and
AAVS1
HSPC from
different sources
N=7,3,5
0 1 2 3 6 Days
FACSAAV6
donor
Nucleases
electroporation
Pre-stimulation
CD34+ cells
CB/BM/MPB
4
12-20 wks after transplant
PB
IL2RG and AAVS1 site
BM analysis
CD34
+ B
Mye
loid
0
20
40
60
80
100
Pe
rce
nta
ge
of
HD
R
with
in s
ub
po
pu
latio
ns
Genovese et al., Nature 2014
Schiroli, ..., Genovese*, Naldini* Science Translational Medicine 2017
Petrillo et al., Cell Stem Cell 2018
• Although improved, the yield of HDR-edited HSPC remain
limiting
– Does it reflect biological response(s) to DNA DSBs impacting long-
term repopulation activity?
Impact of DNA Damage Response on HSPC
G. Schiroli A. Conti
Collab. R. Di Micco
ATMP
p53P
p21
γH2AX 53BP1
Transient
arrest
Senescence
Apoptosis
DSB
Sensors
Apical kinases
Trasducers
Effectors
Senescence-Associated Secretory Phenotype
Milyavsky.. Dick, Cell Stem Cell 2010
Mohrin.. Passegué, Cell Stem Cell 2010
Non-linear dimensional reduction with supervised approach
(gene list from Fares et al., Blood 2017)
Single cell transcriptomic
HSPC Response at Single-Cell Resolution
Schiroli, … Genovese*, Naldini*, Di Micco*; Cell Stem Cell 2019OR 961: Schiroli, May 2nd – Excellent Award
Engraftment of human cells (PB) % GFP in Human PB cells
n=5,6 mice/group
Inhibiting p53 response improves edited HSC fitness
p53 Transcr Signature
0 1 2 3 Days
Pre-stimulation
CD34+ cells
4
(Milyavsky et al., Cell Stem Cell 2010)
CRISPR/Cas9 RNP
electroporation
+/-GSE56 mRNA
AAV6
donor
Schiroli, … Genovese*, Naldini*, Di Micco*; Cell Stem Cell 2019ASGCT OR 961: Schiroli, May 2nd – Excellent Award
• Primary immunodeficiencies such as: IL2RG,
RAG1/2, CD40L,
– provide best risk-benefit ratio for first clinical testing
– unregulated gene expression may pose risk of
transformation or malfunction
– selective advantage of gene corrected progeny may
compensate for low editing efficiency
– lymphoid progenitors may provide therapeutic
benefit in the absence of long-term engrafted HSC
Towards Clinical Testing of Targeted Gene
Editing in HSC Gene Therapy
“One Size Fits all” Gene-correction Strategy
GFPProm
IL2RG locus (X Chr.)
IL2RG-cDNA
ZFNs Target
Sites
Exon
Donor
HDR-mediated Integration
Transcript from
the endogenous
promoter
SASD
GFPPromIL2RG-cDNA
polyA
IL2
RG
GFP+
♂ Lymphoid cells IL2RG -/-JY-_R005
Gated on: Gate 3
GFP-A
AP
C-A
-102-10
110
210
310
410
5
-102
102
103
104
105
MFI+: 1485MFI-: 1574
47.85%3.35%
48.44%0.35%
JY - Gated on: Gate 3
GFP-A
AP
C-A
-102-10
110
210
310
410
5
-102
102
103
104
105
MFI+: 1220MFI-: 1553
0.27%98.89%
0.02%0.83%
UT
IL2RG signalling pathway analysis
IL-2
IL-15
NSG mice
for SCID-X1
Modeling SCID-X1 Gene Correction in Mice
• Required threshold of functional HSPC to rescue
SCID-X1 disease in mouse model
– 10% of HSPC transplant input
– within reach of optimized protocols & reagents
• Establishes rationale for clinical translation
• Road map for the development of HSPC gene editing
strategies
Schiroli, ..., Genovese*, Naldini* Science Translational Medicine 2017
RAG1: In coll. with A. Villa and L. Notarangelo
Tranplant
Wild-type + SCID HSPC
at decreasing ratioSCID-X1 mice
x
x
HIGM1
Patient
CRISPR
AAV6
T cells
Re-infusion of
autologus
corrected cells
Transplanted
corrected cells
restore the
immune
system
CD40LG deficiency cause HIGM1 syndromeMostly expressed on activated CD4+ T cells
Induces B cell activation and Ig class-switching
T cells well amenable to HDR gene editingHigh safety of the clinical cell product
Eradicate life-threatening infections
Gene Correction of CD40LG on T cells and HSC
HIGM1
Patient
CRISPR
AAV6
HSPC
Re-infusion of
autologus
corrected cells
Transplanted
corrected cells
restore the
immune
system
Gene Correction of CD40LG on T cells and HSC
CD40LG deficiency cause HIGM1 syndromeMostly expressed on activated CD4+ T cells
Induces B cell activation and Ig class-switching
T cells well amenable to HDR gene editingHigh safety of the clinical cell product
Eradicate life-threatening infections
Potentially bridge to HSC Transplant
Vavassori, ..., Naldini*, Genovese*. Manuscript in preparationASGCT OR 656: Vavassori, May 1st – Travel Award
• Giulia Schiroli
• Samuele Ferrari
• Aurelien Jacob
• Valentina Vavassori
• Elisabetta Mercuri
• Luisa Albano
• Martina Fiumara
• Angelo Lombardo
• Anna Villa
• Genni Marcovecchio
• Nicolò Sacchetti
• Carmen Castiello
• Eugenio Scanziani
• Raffaella Di Micco
• Anastasia Conti
• Ivan Merelli
• Stefano Beretta
Acknowledgments
• Luigi Naldini • Philip D. Gregory
• Michael C. Holmes
• Anthony Conway
• Cecilia Cotta-Ramusino
• Carrie Margulies
• Jennifer Gori
• Vic Myer
• Luigi Notrangelo
We are looking for brilliant post-docs or PhD students