programme lecture introduction to gene & cell therapy for
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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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2
Get Ready for Quizzes…
3
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
4
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
20 20
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)
30 30
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.)
40
Quiz 3
Gene therapy
for the masses?!
Depeche Mode – Music for the Masses
41
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
42
“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
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