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[PDF] EIC-ERC workshop on Gene and Cell Therapy

117031_3NaldiniL.pdf

Genetic Engineering of

Hematopoiesis

to Treat Inherited Diseases and Cancer

Luigi Naldini, MD, PhD

EIC-ERC workshop on

Gene and Cell Therapy

29 June 2021

A New Medicine for the 3rdMillenium

New technologies for transferring and

editing genes (Gene Therapy)

Effective strategies to isolate and

transplant stem cells (Cell Therapy)

Improved manipulation of biological

weapons of immunity (Immunotherapy)

Make possible to design new therapies

for diseases until now without treatment

A New Medicine for the 3rdMillenium

New technologies for transferring and editing genes (Gene Therapy)

Gene Replacement

Lentiviral Vectors

Gene Editing

ZFN

CRISPR/Cas

Emerging Break-free Editors

Base Editors

Prime Editor

Epigenetic Editing

1995 00 2005 2010 2015 2020

Salk Institute, 1995

Achieveefficient& stablegene transfer

in stemcells(ex vivo) or long-livedtissues(in vivo)

Regulatetransgeneexpression

Ectopicor constitutiveexpressionmaybe toxic

Avoidimmune response

Mayneutralizetherapyand cleartransducedcells

Alleviate vector-relatedtoxicity

Inflammatory response to in vivoadministration

Risk of insertional mutagenesis: integrationmay

activateoncogenes, disrupttumorsuppressorgenes

Off-target DNA breaks, translocations in editing

Challengesto Safe& EffectiveGene Therapy

A New Medicine for the 3rdMillenium

New technologies for transferring and editing genes (Gene Therapy) Effective strategies to isolate and transplant stem cells (Cell Therapy) exploiting their regenerative potential

Blood Stem Cells (Hematopoietic Stem Cells HSC)

Skin & cornea

Muscle

Bone

HarvestHematopoietic

StemProgenitor Cells

Peripheral blood

DendriticCells

Microglia

Macrophages

Monocytes

Red Blood Cells

Granulocytes

Platelets

Thymus

Gene

Transfer

or Editing

Cancer, Infections

Tissues

Infuse into

conditioned recipient

Immuno-hematologicaldiseases

T Lymphocytes

NK Cells

B Lymphocytes

Storage diseases

Clinical Testing of HSC Gene Therapy

Pioneering work with -RV in PID

ADA-SCID 1stGT on EU market (Strimvelis)

Expanded applications with Lentiviral Vectors

Up to 90% stable marking

No genotoxicity to date

Tiget trials: 120 pts, up to 10 yrfollow up,

persistent clear clinical benefit

Metachromatic Leukodystrophy, Wiskott-

Aldrich Syndrome, -thalassemia, MPS-I

2 therapies already approved for EU market

(Zynteglo, Libmeldy) with GSK/OTL

Similar findings in several other trials & sites

~350 pts; up to 19 (11 for LV) yrfollow-up

ALD, X-SCID, Sickle CD, CGD, MPS-IIIb,

ĂŶĐŽŶŝ͕ĂďƌǇ͙

Hematopoietic Stem Cell (HSC) Gene Therapy

HSPC Rationale for HSC GT of MetachromaticLeukodystrophy

Afterconditioning, some microglia

reconstitutionfrom infusedprogenitors

ĺEnzyme overexpression

Cross-correctionof

residentcells

Blood

CNS Biffi et al., J. Clin Inv. 2004 and J. Clin Inv. 2006; Capotondo et al., PNAS 2012

Arylsulfatase A, ARSA

GALACTO

CEREBROSIDESULFATIDE

Geneticdeficiencyof ARSA

Storage of myelinbyproduct

Degenerationof oligodendrocytes,

microglia and neurons

Severe dismyelination

Prognosis

fatal within10 yearsfrom onset

Therapy

HSC transplantpoorlyefficacious

Clinical Benefit of HSC Gene Therapy in MLD

Licensed to Orchard Therapeutics

Biffi, Montini et al, Science 2013; Sessa, Lorioliet al., Lancet 2016

Fumagalli, Calbi et al. in revision

In vitroIn vivo

LV -RV

Genotoxicity

2,1601,3421,7497479454,89910,680

0103060912182430364248546066

100
PB % 0

Lentiviral

Trial

(Milan)

WAS gRV, patient 9

030710131720232731373839

Months after GT

100
% 0 * MECOM -Retroviral Trial (WAS, from Braun et al., Sci. Transl. Med. 2014)

Start SiteGene

Where does it insert?

How many clones?

Are they expanding?

Which genes are targeted?

Vector Integration Site Analysis

Genotoxicity

2,1601,3421,7497479454,89910,680

0103060912182430364248546066

100
PB % 0

Lentiviral

Trial

(Milan)

Where does it insert?

How many clones?

Are they expanding?

Which genes are targeted?

David P. Steensma et al. Blood 2015;126:9-16

Age-related

Clonal Hematopoiesis

Chemotherapy-related

Gene therapy-related?

Autologous HSC GT may become

preferred to allogeneicHSC transplant in genetic diseases

Available to every patients

Abrogates risk of graft vs. host disease &

rejection

Mixed chimerism sufficient for full benefit

Enhanced benefit by increased gene

dosage

Beyond gene replacement

Target delivery of biotherapeutics at

disease sites

CNS via microglia progenitors

Outstandingchallenges

gene transfer rate sometimes limiting and variable among patients genotoxic conditioning delayed rate of engraftment

Concerns(long-term)

residual genotoxic risk long-term stability of transduced cells graft

Expectedimprovements

increasedHSC input (improvedharvest, ex vivo HSC expansion) milderconditioningregimens (non mutagenic)

A Future Outlook for HSC Gene Therapy

A New Medicine for the 3rdMillenium

New technologies for transferring and

editing genes (Gene Therapy)

Effective strategies to isolate and

transplant stem cells (Cell Therapy)

Harness biological weapons of immunity

(Immunotherapy) direct them against tumor cells or pathogen infected cells

Engineer T-cell specificity

against tumor

TCR/CAR gene transfer

Cell & Gene Mediated TumorImmunoTherapy

Hurdles: TCR dilution& misparing, competitionwithCAR

Poorlyeffective& potentiallyautoreactiveT cells

ĺDisruptendogenousTCR genes

Generatenew T CellReceptor againstTumor Antigen

Isolatedfromraretumor-infiltratinglymphocytes(TCR)

Builtas artificial ChimericAntigenReceptor (CAR)

T cells from patient

TCR gene transfer

Nano- Gene

Knock Out

DNA DSB

at target site

TALENs

CRISPR/Cas9ZFNs

Genomic DNARepair by

Non Homologous

End Joining

Loss of Function

Loss of nucleotides

TRBCZFNs

Disrupting TCR Genes in Lymphocytes

TRBC1TRBC2

ZFNs site

TRBD2TRBJ2TRBD1TRBJ1

ZFNs site

TRBV x 52x 6x 7

TCR locus

(Chr. 7q34)

Genetic Editing of TCR Specificity

WpreRREGATCR TCR

Provasi et al, Nat Medicine, 2012; Casucci et al, Blood 2013; Mastaglio et al, Blood 2017

CART & TCR transfer

Remarkable efficacy in some

tumors(ALL, Lymphoma)

Ongoingimprovements

Enhancedactivity

Editing out checkpoint controls

PhysiologicalExpression

Improvedspecificity&

bettercontrol of toxicity

Switches, conditionalsuicide,

syntheticcircuitry

Allogeneicsource

off-the-shelfproduct

Poorlyeffectiveon solidtumors

Immunosuppressive tumor micro environment

limits recruitment & activity

Adoptive Cancer Immunotherapy

T cells: limited reactivity to

Tumor Antigens

Inhibited effector function,

TregTh2 skew

Tumor-Associated

Macrophages (TAM)

promote angiogenesis, tissue remodeling, invasion, immunosuppression

Parker et al., 2016

Tumor-targetedGene Deliveryof Immuno-activatingCytokines

Stable expression targeted

to disease sites

Sustained local concentration

within therapeutic window (no peak & troughs)

Preventing off-target effects

& reducing toxicity

May prevent de-sensitization

& counterregulatory effects

PleiotropicImmunoactivation

by IFN-

ȴϯ

IFN-ɲ

2xmirT126

WPRERREGA

ȴϯRU5R

SASD Tie2P cPPT

Tie2 E

Hematopoietic

stem cell TIE2+ miRNA126+

IFN-OFF

Myeloid

stem cell

MonoblastPluripotent

stem cell

Tie2+

Monocyte

Tie2+

Macrophage

TIE2+ miRNA126-

IFN-ON

LV HSCT

Control (Empty vector)

Tie2-Ifna1-mirT

GBM inoculation

(500,000 cells)

C57Bl/6 mice

Lethally irradiated

0 6 Donor HSPC

Weeks of age

MRI >13 Tumor-targetedGene Deliveryof IFN-by TAM InhibitsGBM

Tumorsite

Escobar et al., Sci. Transl. Med2014 & Nat. Comm 2018; Birocchi, Coltella et al., submitted

Translating IFNaGene Therapy to the Clinic

IFN-LV

transduced HSC

Harvest HSPC from

GBM patients

Re-infuse transduced HSPC with

unmodified HSPC after (sub) myeloablative chemotherapy

Unmodified HSC

Diagnosis

Methylated

Non-

Methylated

Not

Eligible

Enrolment &

Screening

HSPC mobilization & apheresis

Radiotherapy

(SOC)

Infusion of non-

manipulated HSPC & Temferon

Follow-up phase

2 years

Temferon production & release

Conditioning

Day 0

TEM-GBM_001 study (NCT03866109)

SurgeryMGMT

P.I.: G. Finocchiaro(Besta)

F. Ciceri (OSR)

Extension

Study

(N= 6 patients)

Phase I/IIa

single-center, open-label dose escalation study

Gene Therapy 2.0: Gene Editing

restores gene function and expression control genotoxicrisk circumscribedto off target activity challengingto comprehensively define potentialfor translocations, large deletionsand bi-allelichits constrained in HSC by low efficiency of HDR need for DNA template codelivery

Genomic DNARepair by

Homologous

Recombination

mutation

Gene Correction

Homologous

Template

with correct sequence

Genomic DNA

DNA DSB

at target site

TALENs

CRISPR/Cas9ZFNs

Gene correction

ATM P p53 P p21

ȖH2AX53BP1

Transient

arrest

Senescence

Apoptosis

DSB

Sensors

Apicalkinases

Trasducers

Effectors

Inflammatory cytokines (SASP)

+ GSE56

ĹnumberĹgrowth

81218
0 20 40
60

Weeks after transplantation

# dominant unique BARsRNP + AAV6 + GSE56

05101520250

20 40
60

80HS/AAV6

HS/AAV6+GSE56

Weeks after transplantation

% hCD45+ cells ****

RNP + AAV6

+ GSE56

Optimizing HSC Gene Editing

Schiroli, Conti et al, Cell Stem Cell 2019; Ferrari, Jacob et al., Nat Biotech, 2020

Primary immunodeficiencies

IL2RG, CD40L, RAG1/2

unregulated gene expression pose risk of transformation or malfunction selective advantage of corrected progeny compensates for low editing efficiency

Suitable risk-benefit ratio

Schiroli et al, Sci TranslMed2017

Vavassori, Mercuri et al, EMBO MolMed2020

TowardsClinical Testing of HSC Gene Editing

DNA DSB at target site

NHEJ

Non Homologous

End Joining

Gene Disruption

G1S/G2

Exogenous

donor template

Gene Correction / Transgene Insertion

HDR

Homology Driven Repair

CRISPR/Cas9Sickle Cell disease, -thalassemia

rescue of fetal hemoglobin by disruption of -globin repressor expression (erythroid enhancer)

FrangoulCorbacioglu; N EnglJ Med. 2021

Summary: Choosing the Right Tool

Lentiviral Vectors

for Gene Replacement

Genome-wide insertion

Variable expression level

Highly Efficient

Clinically tested

Compatible with long-term HSC

Residual insertional genotoxicity?

Nuclease-based Editing

for Gene Correction

Targeted insertion

In situ reconstitution

Constrained in HSC

Early clinical stage

Impact on long-term HSCTBD

Off-target activity,

large genomic rearrangement?

Base (and Prime) Editors

for Correcting Mutations

Single/few base edit

DNA Break-less/free

Efficient

R&D stage

Unknown

Sequence-independent

off-target activity?

An Evolving Pharmacology

Small drugs

Biologicals

Genes

Stable sustained expression

Targetedand regulated

An Evolving Pharmacology

Small drugs

Biologicals

Genes

Stable sustained expression

Targetedand regulated

Cells

Regenerative potential of

stemcells

Killer action of Lymphocytes

CD8

T cells

Cell & Gene Therapy: the Challenges Ahead

Biology

Still limited understanding of stem cell and

immune regulation

Need to overcome:

innate responses to exogenous nucleic acids and DNA breaks biological constraints to survival, engraftment or regeneration imposed by disease immune barriers to gene transfer & cell transplant evasive resistance to immune therapies in cancer progression

Safety

long-term effects

Bedside delivery

Need for multidisciplinary expertise

Society

Personalized medicine manufacturing

marketing pipeline costs and sustainability

Fair equitable access

Emerging potential for

Germlinegene editing

Although strongly

discouraged for technical, scientific and ethical reasons, it has already been attempted

Currently prohibited in

most countries & under review by international agencies for further recommendations

Somaticgene therapy

managed under existing ethical norms and regulatory regimes for advanced therapies

Limited to treatment of

severe disease or disability evaluated in the context of risks and benefits as other medical treatments enhancement of human capacities discouraged or unacceptable at this time