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

Pietro.Genovese@hsr.it

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