Gene -Therapy technologies for SCD : from no treatment to an era of - - PowerPoint PPT Presentation

‘Gene-Therapy technologies for SCD : from no treatment to an era of too many choices’ ____________________

Sandeep Soni, MD

• Clin. Associate Prof. of Pediatrics
• Div. of SCT and Regenerative Medicine

Lucile Packard Children’s Hospital Stanford University, CA

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Objectives

• 1. Provide an overview of the field with focus on ex-vivo

modification of HSPCs

• 2. Compare and contrast gene-addition and gene-editing

technologies

• 3. Update on Stanford initiative

2

Sickle Cell Disease- Paradigm shift

“Rather than an ‘episodic’ disease, SCD is a chronic inflammatory state leading to continuous organ damage, poor QoL and decreased life- expectancy’’

• Increased need for curative options
• Early intervention

Autologous Gene-Therapy

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Allogeneic HSCT Autologous Gene Therapy Toxicity: conditioning + immunosuppression Toxicity: related to intensity of busulfan Immunosuppression required None Risk of immune-mediated rejection None GvHD No risk Donor availability No donor required Long-term risks: organ toxicities Potential risk of oncogenesis or ‘off-target’ activity

• Gene therapy is a promising approach with many potential benefits

Conditioning

Overview of the treatment plan

Mobilization Infuse

Apheresis

(Plerixafor)

Select CD34+ cells Gene manipulation Cryopreserve, test and release

Subject Treatment Centralized Manufacturing 2 years follow-up

Long term Follow up

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Busulfan myeloablation

Modified CD34+ cells

Soni S, et.al. ASBMT

Autologous Gene Therapy Platform: ‘ex-vivo modification of long term repopulating HSC- one time treatment’

• 1. Correction of LT-HSC
• 2. Gene Modification: Hb

expression under control of a erythroid promoter (lineage specific)

Gene Therapy is ‘in-vogue’

• Technological advancements:
• Whole genome sequencing
• Vectors: more efficient and safe (Lentivirus versus Retrovirus)
• Large payload e.g. HBB gene (15.8 kD)- can be inserted in the vector
• Selection of long term repopulating stem cells (CD34+; Milteyni)
• Efficient transduction of stem cells (small molecules)
• Availability of Gene-editing ‘nucleases’: makes gene editing precise
• Large scale manufacturing
• Short term Safety established
• Favorable regulatory environment: Orphan disease designation; RMAT

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Approaches to correct HSC for SCD

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bluebird bio UCLA Univ.of Cincinnati TIGET, Milan Boston Children’s Stanford UCSF Crispr Tx

• St. Jude’s/Editas

Sangamo

Adapted from Blood, Feb,2016,127

Gene Addition: LentiGlobin BB305: Rationale in SCD

▪ Provide high level βA-T87Q-globin ▪ βA-T87Q-globin incorporates an anti- sickling amino acid substitution also found in γ globin1 ▪ βA-T87Q-globin and γ globin inhibit HbS polymerization2,3

• 1. Takekoshi & Leboulch, PNAS 1995; 2. Ngo et al., Br.J.Haematol 2012; 3. Pawliuk et al, Science 2001

destabilization

bS bA-T87Q

• r γ

polymerization

Phe Val

6

bS bS

Val

6

Gln

87

Thr

87

Leu Phe Leu

9 Soni et al. 2016 BMT Tandem Meetings

Post-transplant Hb fractions

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1301 1303 1204

10 10 15

Months post drug product infusion Hb g/dL

1 2 3 6 9 12 10.9 12.0 10.6 11.4 11.7 9.1 8.5 7.6

5.5

10

8.6 7.8 9.2 8.5

HbAT87Q HbF HbA2 HbS HbA

(post-transfusion)

1.0 0.3

8.6

49%

Anti-sickling Hb

16%

Anti-sickling Hb

17%

Anti-sickling Hb

Data as of 10 Nov 2015 (HGB-205) / 17 Nov 2015 (HGB-206); Data reported from an ongoing trial with an open database

Improving Outcomes: Lessons learnt Potential Ways to Improve Cell Dose, Transduction and Engraftment

11 Kanter et al. 2016 ASH Meeting

1 2 3 2 4 6 6 9 1 2 1 5 1 8 2 1 2 4 M o n t h s P o s t D r u g P r o d u c t I n f u s i o n H b A

T 8 7 Q C o n c e n t r a t i o n ( g / d L )

G r o u p B 1 3 1 3 G r o u p A G r o u p B 1 3 1 2

H G B - 2 0 5

Improvements in Drug Product Characteristics and Protocol Improve HbAT87Q Production

Higher DP VCN Higher in vivo VCN Higher T87Q

• 51% HbAT87Q
• Total Hb 12.6

g/dL NEJM patient

N=9 patients 4 patients >6 months FU HbAT87Q 4.8-8.8 g/dl Like Sickle Cell Trait

Safety of the Lentivirus vectors for gene insertion

▪ Semi-random insertions: ‘ safe-sites’ for insertions? ▪ No Leukemia reported ~250 patients treated with lentivirus vector based GT in the last 5 years ▪ 1 patient in trial has developed MDS ▪ No RCL (HIV infection) till date ▪ FDA requires 15 years follow-up

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HbF production is controlled by a genetic ‘switch’ in stem cells

HbF Re-expression Strategies For Hemoglobinopathies

Prevent Repressor expression

(BCL11A enhancer disruption)

x

Block Binding

(recreate HPFH deletions)

x

Chr 2 Chr 11

Interfere with BCL11a mRNA

BCH approach: RNAi technology

Lentiviral vector to deliver shRNA segment to inhibit the BCL11a mRNA Block BCL11a in erythroid progenitors; inhibitory-RNA tagged to HBB promoter (erythroid specific) Goal to increase Hb F production in red cells N=4 patients enrolled First patient: F-cells (>25% HbF)- 80% in peripheral blood Resolution of symptoms BCL11a : a good target to increase HbF

Gene Editing Tools- Ability to make precise cuts

Precise Spatial Modification (DSB stimulates process by >1010) Precise Spatial AND Nucleotide Modification of Genome (DSB stimulates process by >105) ZFNs TALENs HEs CRISPR/Cas9 (class)

Donor DNA

*

Homologous Recombination (copy and paste) Non-homologous end-joining (stitching)

Mega-Tal

DNA Scissors Advantages of Crispr-Cas9

1. High efficiency (in-del) 2. Precise DSB (less off-target) 3. Conserves endogenous promoters and regulatory elements

• 4. Easy manufacturing/cost

Pros and Cons of gene-insertion versus gene-edit

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Parameter Gene Addition Gene- editing Insertions Semi-random Precise edits MOA Produce HbA or F Recreate HPFH Delivery Lentivirus vector transduction Nuclease or Cas9+ sgRNA Extrapolation Regulation of gene expression Vector to provide promoters and regulatory elements Uses endogenous regulation Efficacy Transduction efficiency In-del efficiency,

• r HDR efficiency

Safety Recombinant HIV no Insertional oncogenesis ‘off-target’ activity Cost High Low

Each Nuclease- Variable

• 1. Extrapolation capability
• 2. Efficiency
• 3. Precision (‘off-target’)

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Crispr Therapeutics Approach: Results of editing in healthy donor stem cells

Soni,S. pre-clinical development

Disrupt the BCL11a enhancer region by Crispr-Cas9 editing Research: optimization Goal: effective and safe Highly efficient and precise; Trial is open for enrollment at multiple centers

Stanford Approach

Cas9/gRNA (100 nt) complex (RNP) Donor DNA (AAV)

Homologous Recombination

Editing E6V Sickle Mutation in Multiple SCD Patients

HbS/HbS INDEL/HbS INDEL/INDEL HbA/HbS HbA/INDEL HbA/HbA 10 20 30 40

% of Methyl Clones

60 clones (3 patients) 49% HR

S/S

Sickle INDELs HR 10 20 30 40 50 60 70 80 % alleles

Allele Frequency in Human Cells at 16 Weeks Post- Transplant into NSG Mice Allele Frequency in CD34+ HSPCs Prior to Transplant into NSG Mice

High Frequencies of Gene Correction at HBB in Patient Derived CD34+ HSPCs that is maintained after transplantation into NSG mice

Approaches to correct HSC for SCD

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bluebird bio UCLA Univ.of Cincinnati TIGET, Milan Boston Children’s Stanford UCSF Crispr Tx

• St. Jude’s/Editas

Sangamo

Adapted from Blood, Feb,2016,127

Key Takeaways

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• Multiple gene-insertion and editing approaches

‘targets and tools’ available

• Long term benefit and safety are the key issues

All clinical trials are fairly new

• Crispr-Cas9 approach: easy manufacturing, high

efficiency and precision of editing, minimal toxicity in hHSC

• Stanford Gene-editing Trial start by early 2020

How do I choose which technology is better?

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• 1. All are experimental trials
• 2. All are intended to ‘ameliorate’ ongoing organ damage
• 3. Risks are different for each technology
• talk to your doctor/hematologist
• analyze the risks: short term versus long term
• 4. Is waiting the right strategy?

Participation in Clinical trials benefits patients and society It’s only the brave that change the world

Questions

Rate Limiting Steps and Future

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Rate Limiting Steps:

• 1. Manufacturing
• 2. Regulatory
• 3. Development of Assays
• 4. Cost of trials

Future:

• Competitive field
• Small number of patients
• Costs?
• Risks of Leukemia / off-target

Benefit to patients

Confidential - 28

Val 6 Phe 85 Leu 88

Lateral Contact

Val 6 Val 6 Phe 85 Leu 88 Phe 85 Leu 88

87 100 g-globin HLDDLKGTFA Q LSELHCDKLHVDPENF | | | : | | | | | | | | | | | | | | | | | | | | | | b-globin HLDNLKGTFA T LSELHCDKLHVDPENF | | | | | | | | | : | | | | | | | | | | | | | | | | d-globin HLDNLKGTFS Q LSELHCDKLHVDPENF V F L T V F L Q polymerization destabilization

b1 a1 b2 a2 a1 a1 a2 a2 b1 b2 b2 b1

Axial Contact

Antisickling Properties of βA-T87Q-Globin Chain

Confidential - 29

How Much Expression of Antisickling Globin is Needed for Clinical Effect?

• Subjects who are compound heterozygotes for SCD and hereditary

persistence of fetal hemoglobin are typically asymptomatic

• If 1g of therapeutic globin is the functional equivalent of 1g of fetal globin,

these data argue that as little as 3g/dL of therapeutic globin could have significant clinical benefit

Ngo et al., 2011

Goal – Hb A production and reciprocal decrease in HbS : convert patients to trait

Mechanism of Action of β-globin Gene Addition: Lentivirus Vector

Effect of Gene addition on sickle cell

βSβS γγ βA-T87Q production 1. Reciprocal decrease in HbS (Quantitative)

• 2. Improved RBC survival

3. HbAT87Q resists polymerization: reduction in sickling (Qualitative) αα/αα

RBC survival advantage leads to higher level of correction in the peripheral blood compared to BM (similar to allogeneic HSCT)

Confidential - 31

Efficacy of BMT and Gene therapy for SCD

SCD BM Mixed chimerism post –BMT 50% Normal peripheral RBC HbA predominates SCD BM Engraftment of GT cells Peripheral- Hb S trait Random and variable insertions of Lenti-G gene into HSC HSC SS

BMT GT RBC survival advantage RBC survival advantage

‘’Pan-cellular’’ correction

Variables-

1. Transduction efficiency 2. % cells marked 3. Myeloablation 4. Inhibition of polymerization

HbT87Q 50% Hb S 50% ‘’Hetero- cellular’’ Correction Assessment of SS, A/S, AA RBCs ? HbA 80% Hb S 20%

1 copy 2 copies 3 copies

Confidential - 32

What we do – Manufacture Virus With a Gene Payload

Large Scale production GMP BB305 Lentiglobin