Carsten W. Lederer, PhD Molecular Genetics Thalassaemia DepartmentThe Cyprus Institute of Neurology and Genetics & The Cyprus School of Molecular Medicine

Lederer@cing.ac.cy

Introduction to Gene & Cell Therapy

for Haemoglobinopathies

The Cyprus Institute of Neurology and Genetics

3 August 2020Virtual Teaching

Programme Lecture

ARISE African Research and Innovative

Initiative for Sickle Cell Education

Outline

Gene Therapy

Principles

Tools

Gene Therapy for Haemoglobinopathies

Key Molecular Players

Diseases and Options

Application Examples

Cypriot Studies

Trials and Tribulations

Gene Therapy for the Masses?!

Accessibility of technology

“Catching them early”

Current Trends

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Get Ready for Quizzes…

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1. Go to: https://b.socrative.com/login/student/

[or: socrative.com > Login > Student Login]

2. Enter Room Name: CWL

3. Enter Your Name/Identifier

I will initiate/evaluate the quiz during the presentation.

Gene Therapy

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Principles: Overview

Introduction of genetic material to heal or prevent disease

Strategies Cell repair

(of defective or injured cells)

Cell replacement

(of defective, injured or dead

cells) with corrected cells [Cytotoxicity (to remove

or weaken deleterious cells)]

Execution dependent on disease mechanism Ex vivo correction, e.g. of autologous stem cells

and re-implantation Ex vivo (induced) or in situ (spontaneous) differentiation into target cell types

In vivo correction of gene defects in differentiated or stem cells

Free PMID 25091489 and 25227756 5

https://www.ncbi.nlm.nih.gov/books/NBK538378

Principles: Some Risks and Benefits

Advantages Potentially curative (if permanent)

Potentially universal (independent from compatible donors)

Downsides for inherited disorders Stable, insertional or editing approaches

Permanent, but with cancer risk (by insertional mutagenesis)

Save, non-insertional approaches Usually not permanent

(by loss of epigenetic elements)

Current high cost of genetic medicines, owing to Stringent QC requirements for GMP

(clinical-grade) reagents

Limited production capacity

(Previously) long development time

XX

X

X

X

X

X

X

Indel/LV

Tumour suppressor gene

Proto-oncogene

Indel/LV

Indel/LV

Indel/LV

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Gene addition (aka gene augmentation) Addition of functional gene copy

For permanent cure, integration is required

Gene editing (correction, disruption, excision) DNA-level correction of defects

Faithful correction is inefficient, disruption is highly efficient

Originally based on double-strand break (DSB) repair

Ongoing development of DSB-independent editors (epigenetic editors, base editors, prime editors)

Principles: Addition vs Editing7

cPPT μLCRp LTRLTR βpIIIIIIRRE

Tools: Gene Addition

Δ

e.g. β-globin

www.addgene.org

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Example: lentiviral vectors as transfer vehicles in vivo and ex vivo

Biosafety

Based on HIV

Disarmed and replication-defective

Compartmentalised

Design considerations

<8.5 kb capacity

Achieving physiological expression

Tools: Gene Editing

Finotti et al. (2015) Journal of Blood Medicine PMID 25737641Patsali et al. (2019) Molecular Diagnosis & Therapy PMID 30945167 (Disruptive Technology)

Papasavva et al. (2019) Molecular Diagnosis & Therapy Free PMID 30945166 (Rare Opportunities)

ideal for disruption & tagging

DSB

FokI

FokI 5’3’5’

Right TALEN

Left TALEN TALEN

3’

Precise protein code

5’3’5’

3’

FokI

Left ZFN

Right ZFN

ZFN

FokΙ

Approximate protein code FokI

NHEJ 5’3’5’

3’

Disruption

3’3’

gRNA

CRISPR/Cas9 (RGEN)

5’PAM

5’ AGGGUACAGUCACAUUCAGAU 5’Cas9

RNA-DNA basepairing NGG

3’5’

5’3’5’

3’

RepairHDR

required for precise (ORF) editing

PUBMED citations 9

Tools: Base Editors

gRNA5’ AGGGUACAGUCACAUUCAGAU

mediates recognition

Targeted base conversion, stimulated by nick, without DSB

Cytosine > Uracil (read as thymine; by cytidine deaminase)

Adenosine > Inosine (read as guanine; by artificial adenine deaminase)

Employing engineered single protein based on mutated Cas9

Fix missense mutation (repair) or introduce non-sense mutations (stop)

C

Rees et al. 2017 Nature Communication PMID 28585549

Li et al. 2017 Protein Cell PMID 28825190

Wang et al. 2017 Cell Research PMID 28849781

Cas9Cas9

uracil DNA glycosylase inhibitor

prevents removal of base edit

cytidine deaminase

CU conversion in ssDNA loopout within 2–5 bp

creates ssDNA loopout to allow base conversion

nicks & prompts mismatch repair of non-target strand

CCyD

non-target DNA strand

target DNA strandUGI

U

Gaudelli et al. 2017 Nature PMID 29160308

Kim et al. (2017) Nature Biotechnology PMID 28191901

Plosky et al. 2016 Molecular Cell PMID 27203175

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Quiz 1

Gene Therapy for Haemoglobinopathies

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Key Molecular Players

α β

β α

HbA (>95%)HbA2 (<3%)

α δ

δ α

HbF (<1%)

α γ

γ α

α1 α2ζα-globin locus Chr. 16

ε Gγ Aγ δ ββ-globin locus Chr. 11

HbF (>20%)

α γ

γ α

α β

β α

HbA (>75%)

MYBKLF1

GATA

1FOG1

NuRD BCL11ANuRD LRF

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γ-globin as positive modifier of β-haemoglobinopathies

BCL11A as repressor of γ-globin and potential therapeutic target

Erythroid-specific suppression of BCL11A expression

RNAi of BCL11A using erythroid-specific shRNA expression

Disruption of erythroid-specific transcriptional enhancer of BCL11A

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γ

α β

β α

βS

βS

α

α

βS

βS

α β

β α

α

α

β-thalassaemiaSCD

MYBKLF1

GATA1FOG1

NuRD BCL11ANuRD LRF

ε Gγ Aγ δ β

CFU-TL/BL

BCL2 MDM2

p53

CFU-E

β-globin

BCL11A mRNA

BCL11A-XL//

Diseases and Options: γ-Globin Induction

CFU-TL/BL

p53 apoptosis

CFU-EBCL11A mRNA

γ-globin

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Recessive and potentially lethal disorders

1 SCD mutation, >400 β-thalassemia mutations (β++ – β0)

Homozygous and compound heterozygous disease causation

β-Hemoglobinopathies15

α β

β α

α

α

Removal of

excess α?

Anaemia

persists.

Addition of

β?

Curative.

Repair βmut?

Curative.

Activation of

β-like γ-

globin?

Curative.

β-thalassemia sickle cell disease

α β

β α

βS

βS

α

α

βS

βS

Removal of

βS?

Anaemia

persists.

Addition of γ

or βanti-S?

Curative.

Repair βS?

Curative.

Activation of

β-like γ-

globin?

Curative.

Addition of γ

or βanti-S?

Curative.

Activation of

β-like γ-

globin?

Curative.

Addition of γ

or βanti-S?

Lucrative.

Activation of

β-like γ-

globin?

Lucrative.

Repair βmut

Curative.

More curative?

Lucrative?

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Diseases and Options: Gene Therapy Approaches

1. Gene Addition – with integrating lentiviral vectorsUniversal for SCD and β-thalassaemia

E.g. addition of β-like globins or shRNAs

2. Gene Correction – with designer nucleases or base editorsMutation-specific

E.g. mutation-specific precise repair

3. Reactivation of γ-globin – with various tools and approachesUniversal for SCD and β-thalassaemia

E.g. deactivation of γ repressor BCL11A

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Correction of HBBIVS I-110(G>A)

Deactivation of BCL11A-XL etc.

Current Clinical Trials

Combination therapy with RNAi

or

?

Addition

of “β”

Curative.

Activation

of γ

Curative.

Repair

of β

Curative.

Focus of current development

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Diseases and Options: Ex vivo Gene Therapy17

corrected stem cells(the ‘drug product’)

(2)

conditioning(chemotherapy)

HBB LV

vector production

(1)

(6)

isolation of stem and progenitor cells

affected stem cells

Safety?Efficacy?

Volunteers.Informed Consent.

editor/nuclease

transduction

preincubation(3)

in vitro differentiation

(4)

re-engraftment

(5)

transfection

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Quiz 2

Application Examples

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Application Examples Cypriot Studies

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IVS I-110 β-thalassaemia21

Intronic mutation of β-globin (HBB)

Creation of aberrant splice acceptor site (GG>AG)

Partial dominance: reduced β-globin from normal loci

intronic sequence

Exon 1 Exon 2

ORF

Exon 1 Exon 2

Exon 1 Exon 2 Exon 3

+1 normal SD +131 normal SA

+110 aberrant SA

10% of the β-globin levels of healthy controls

Degradation

60% aberrant mRNA

40% normal mRNA40% normal mRNA

10% of the β-globin levels of healthy controlsORF

trans action of aberrant HBB mRNA?

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IVS I-110 β-thalassaemia22

HBBIVS I-110

Relative carrier

frequency: 75.9%

Absolute carrier

frequency: 9.1%

Kountouris et al. (2014) PLoS1 PMID 25058394

Kountouris et al. (2016) Scientific Reports PMID 27199182 http://www.ithanet.eu/db/ithamaps

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Application ExamplesLess is more

RNAi of HBBIVSI-110(G>A)

aberrant RNA

GLOBE HBB LV

1 2 3LCR βp

U6p Anti-HBBIVSI-110(G>A)

RNAi LV

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Less is more

Patsali et al. 2018 Haematologica PMID 29700171

U6p Anti-HBBIVSI-110(G>A)

RNAi LV

shRNA recognition sites

aberrant HBB mRNA

MEL-HBBIVS

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Patsali et al. 2018 Haematologica PMID 29700171

Less is more

Mock Scr Mid GLOBE GLOBE & Mid

** ******* ** * **** ****

GLOBE HBB LV

1 2 3LCR βp+

HBBIVSI-110(G>A) CD34+

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Application ExamplesDARE

Disruption of Aberrant Regulatory Elements for

HBBIVSI-110(G>A)

HBBIVS1-110(G>A)

1 2 3*

TALEN RGN

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**** ****

DARE – HBBIVSI-110(G>A)

Patsali et al. 2019 Haematologica PMID 31004018

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DARE – Biosafety

DSBs can be the source of Recombination with the on-target locus

Mutagenesis by off-target editing

Targeted deep sequencing of predicted off-targets and HBD

TALEN RGN

RNF219 AS1

Patsali et al. 2019 Haematologica PMID 31004018

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DARE – Elsewhere

Over 300 known disease-causing mutations in over 100 disorders meet DARE criteria

Wide applicability of the efficient DARE approach

Current developments will make personalized therapies affordable

Primarily affected Exemplary Exemplary mutations

organ system disorders Gene dbSNP ID Effect1 Frequency2

Circulatory Poikilocytic anemia SPTA1 rs757147440 aSA 25%

Endocrine Hyperinsulinemic hypoglycemia ABCC8 rs151344623 aSA 68.8%

Nervous Leber congenital amaurosis CEP290 rs281865192 cSD activation 43%

Sensory Stargardt disease ABCA4 rs1457937638 cSD activation 7.5%

Muscular Miyoshi myopathy DYSF rs1285082850 cSD activation 17 families

Congenital muscular dystrophy FKTN rs1554754182 cSD activation 20.8%

Integumentary Erythropoietic protoporphyria FECH rs2272783 cSA activation 42.6%

Respiratory Cystic fibrosis CFTR rs397508266 aSD 2.0%

Multisystemic Fabry disease GLA rs199473684 cSD activation 41.1%

Cancer Breast cancer BRCA2 rs191253965 cSD activation 0.2%

1

Patsali et al. 2019 Journal of Clinical Medicine, 2019

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Application Examples Trials (and tribulations)

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Key trials – BB Overview

https://clinicaltrials.gov/

Ribeil et al. (2017) NEJM PMID 28249145

Thompson et al. (2018) NEJM PMID 29669226

Cavazzana et al. (2017) Molecular Therapy PMID 28377044

Bluebird Bio (LentiGlobin vectors) based on βT87Q-globin

First authorised human trials with HPV569 vector (“LG001”)

Transfusion independence of 1 out of 3 patients reported

Temporary clonal dominance for HMGA2 insertion event

Shortcomings in vector production, transduction efficiency, stability

Follow-up trials with modified BB305 vector

CMV-driven production and no cHS4 insulator: higher titres & stability

Many subjects with speedy transfusion independence

#NCT01745120 phase-1/2 trial for β-thalassaemia (“HGB-204”, August 2013)

#NCT02151526 phase-1/2 trial for SCD and β-thalassaemia (“HGB-205”, July 2013)

#NCT02633943 long-term (15-year) follow-up (“LTF303” for HGB-204 and HGB-205)

#NCT02140554 phase-1 trial for severe SCD

#NCT03207009 phase-3 trial for β0/β0

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Key trials – BB Details

Mobilisation with G-CSF and plerixafor

Storage of 2x106/kg CD34+ cells as backup, transduction, freezing and testing

Conditioning 4x3.2 mg/(kg*d) iv busulfan, 3 d washout period, infusion

15/22 patients stopped transfusion

(HGB-204, HGB-205)

https://clinicaltrials.gov

Thompson et al. (2018) NEJM PMID 29669226

β+/β0

β0/β0

βIVSI-110/ βIVSI-110

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3 months hypertransfusion

Dynamic dose adjustment

of 4-day conditioning

Key trials – BB Details

https://clinicaltrials.gov/

Thompson et al. (2018) NEJM PMID 29669226

https://www.youtube.com/watch?v=8ecRdm4iGs8, Luigi Naldini

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Increased HbA

production with

increased VCN!

Higher VCN to

achieve transfusion

independence…?

Vectors with better

transgene expression

are the (albeit costly)

solution!

Key trials – Other Gene Addition Trials

https://clinicaltrials.gov/

Cavazzana et al. (2017) Molecular Therapy PMID 28377044

Marktel et al. (2019) Nature Medicine PMID 30664781

Memorial Sloan Kettering Cancer Center (TNS9.3.55 vector)

#NCT01639690 phase-1 trial

Mild conditioning (8 mg/kg busulfan)

No transfusion independence in 4 out of 4 subjects

Trial unofficially stopped (“ongoing but not recruiting”)

Possible future trials with insulated vector

IRCCS San Raffaele (GLOBE vector)

#NCT02453477 phase-1/2 trial

Intraosseous injection, three cohorts of different ages (>18, 8-17, 3-7 years)

10 out of 10 patients treated, greater benefit in younger patients

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Key trials – Other Gene Addition Trials

Boston Children’s Hospital (David Williams BCL11A vector)

#NCT03282656 phase-1 trial

Three cohorts of different ages (18-35, 12-18, 3-12)

Recruiting up to 7 SCD patients

Children's Hospital Medical Center, Cincinnati (Punam Malik γ-globin vector)

#NCT02186418 phase-1 (SCD) / phase-2 (SCA) trial

Recruiting approximately 10 SCD patients (aged 18-35)

Children's Hospital Medical Center, Cincinnati (Donald B. Kohn βAS3-FB vector)

#NCT02247843 phase-1 trial

Estimated enrolment 6 SCD patients (aged 18+)

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https://clinicaltrials.gov/

Key trials – Gene Editing/Disruption

https://clinicaltrials.gov

http://www.crisprtx.com/pipeline

Allife Medical Science and Technology Co. Ltd

#NCT03728322

12 patients, 2 to 60 years of age, not recruiting

CRISPR Therapeutics/Vertex Pharmaceuticals Inc (NHEJ-based disruption of BCL11A

erythroid enhancer)

#NCT03655678 (βThal) and NCT03745287 (SCD) phase-1 trials

45 patients each,

12 to 35 years of age

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Key trials – Gene Editing/Disruption

Virus-free, part-

centralised

procedure

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http://www.crisprtx.com/our-programs/our-programs.php

CRISPR

Therapeutics

(cont.)

Key trials – Gene Editing/Disruption38

https://www.globenewswire.com/news-release/2020/06/12/2047260/0/en/CRISPR-Therapeutics-and-Vertex-Announce-New-Clinical-Data-for-Investigational-Gene-Editing-

Therapy-CTX001-in-Severe-Hemoglobinopathies-at-the-25th-Annual-European-Hematology-Associ.html

CRISPR

Therapeutics

(cont.)

Key trials – Gene Editing/Disruption39

https://www.globenewswire.com/news-release/2020/06/12/2047260/0/en/CRISPR-Therapeutics-and-Vertex-Announce-New-Clinical-Data-for-Investigational-Gene-Editing-

Therapy-CTX001-in-Severe-Hemoglobinopathies-at-the-25th-Annual-European-Hematology-Associ.html

CRISPR

Therapeutics

(cont.)

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Quiz 3

Gene therapy

for the masses?!

Depeche Mode – Music for the Masses

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Accessibility of technology

Lowered bar for translation

Streamlining regulatory frameworks (e.g. GMP requirements)

Incentivising companies by EU & US orphan drug regulation

Falling cost through increasing competition around genetic medicines

Democratisation of research

Away from viral vectors and their costly production

Straightforward design of RGNs

Effective delivery of RGNs as RNPs by nucleofection

GMP in a box

Closed-system prototype as GMP-facility replacement in Fanconi trial

One-off cost of $150,000

1/5 staffing requirement

1/2 processing time

No cleanroom requirements 1 – 2 staff

5 – 10 staffhttps://www.miltenyibiotec.com

html/Adair et al. 2016 Nature Communications PMID27762266

https://www.fredhutch.org/content/dam/public/communications/Photo/2016/10-October/Adair/JennAdairGraphic.pdf

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“Catching them early”

Ramachandra et al. 2014 Frontiers in Pharmacology PMID25566071

Shaw et al. 2014 Stem Cells PMID25186828

Infant gene therapy with incremental benefits

Higher relative stem cell yield

Higher success rate

More substantial correction of disease parameters

Lowered vector requirements

patient from 80 kg to 4 kg vector cost from $100,000 to $5,000

In utero gene therapy as quantum leap

Treatment also of disorders lethal in utero (hydrops fetalis)

Minimal cell and vector requirements (1/1000 of that in adults)

Postnatal: 5x106 cells/kg and 5x108 vector particles/kg

In utero: 3x105 cells and 1.5x107 vector particles total

Direct vector injection possible as outpatient treatment

Pending

Biosafety issues (germline transmission)

Bioethics issues (justification of treatment)

Large-animal studies

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Current trends

Involvement of companies

Orchard Therapeutics (GSK), Bluebird Bio with LentiGlobin

CRISPR Therapeutics, Editas Medicine, Intellia Therapeutics

Universal approaches, e.g. for β-haemoglobinopathies

Induction of γ-globin; anti-sickling β-like globins

Translational research focus

Away from cell lines and even iPS cells towards primary HSPCs

Efforts to boost in vivo long-term repopulation after treatment

Editing & CRISPR/Cas9

New preclinical publications mostly on editing approaches

Editing studies towards CRISPR/Cas9 and non-DSB platforms

Safety

Comprehensive assessment of integration sites and off-targets

Targeted insertion approaches for gene addition

Many studies towards reduced off-target editing

Concerns about pre-existing Cas9 immunity!

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Thank you!

Marina

Kleanthous

Panayiota

Papasavva

Our own work was made possible by funds from the EU 6th and 7th Framework Programmes for research, technological

development and demonstration under grant agreements #26539 (ITHANET) and #306201 (ThalaMoSS), from

Erasmus+, from Telethon of Cyprus and as grants ΥΓΕΙΑ/ΒΙΟΣ/0311(ΒΕ)/20 and EXCELLENCE/1216/0092 from the

Research and Innovation Foundation of Cyprus.

45

Petros

Patsali

Basma

Naiisseh

Nikoletta

Papaioannou

Lola

Koniali

«Thalassaemia Prevention»Thalassaemia Centre & General HospitalΣωτηρούλα Χρίστου Μαρία ΣίταρουΜιχάλης ΧατζηγαβριήλΑνίτα ΚολνάγουΜάριος ΑντωνιάδηςΜιχάλης Αγκαστινιώτης

Cyprus Antianaemia AssociationΜίλτος ΜιλτιάδουςΑνδρέας ΠιερίδηςΝαταλία ΜιχαηλίδουΛοΐζος ΠερικλέουςAll patients and sample volunteers!

«Genome Editing»University of FreiburgToni CathomenClaudio Mussolino

«Advancing Lentiviral Vector»King’s College London, ΗΒMike Antoniou

«ERASMUS+ BLC11A-»Erasmus University Medical CenterSjaak PhilipsenThamar van Dijk