‘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 1
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 • Gene therapy is a promising approach with many potential benefits 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 Potential risk of oncogenesis or ‘off - target’ Long-term risks: organ toxicities activity 4
5 Overview of the treatment plan Infuse Conditioning Mobilization Busulfan Treatment Subject Apheresis myeloablation (Plerixafor) 2 years follow-up Long term Follow up Manufacturing Centralized Modified CD34+ cells Select CD34+ Gene manipulation Cryopreserve, test cells and release 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 7
Approaches to correct HSC for SCD Stanford UCSF Boston Children’s Crispr Tx St. Jude’s/ Editas bluebird bio Sangamo UCLA Univ.of Cincinnati TIGET, Milan Adapted from Blood, Feb,2016,127 8
Gene Addition: LentiGlobin BB305: Rationale in SCD b A-T87Q b S b S b S or γ ▪ Provide high level β A-T87Q -globin Phe Phe ▪ β A-T87Q -globin incorporates an anti- sickling amino acid substitution also Val 6 Thr 87 Val 6 Gln 87 found in γ globin 1 Leu Leu ▪ β A-T87Q -globin and γ globin inhibit polymerization destabilization HbS polymerization 2,3 1. Takekoshi & Leboulch, PNAS 1995; 2. Ngo et al., Br.J.Haematol 2012; 3. Pawliuk et al, Science 2001 Soni et al. 2016 BMT Tandem Meetings 9
Post-transplant Hb fractions 15 12.0 11.7 11.4 10.9 10.6 10 8.6 49% 1204 Anti-sickling Hb 5.5 0 9.1 8.6 10 8.5 7.6 Hb g/dL 16% 1303 Anti-sickling Hb HbA 1.0 0 (post-transfusion) 9.2 HbS 10 8.5 7.8 HbA 2 17% HbF 1301 Anti-sickling HbA T87Q Hb 0.3 0 1 2 3 6 9 12 Months post drug product infusion Data as of 10 Nov 2015 (HGB-205) / 17 Nov 2015 (HGB-206); Data reported from an ongoing trial with an open database 10
Improving Outcomes: Lessons learnt Potential Ways to Improve Cell Dose, Transduction and Engraftment Kanter et al. 2016 ASH Meeting 11
Improvements in Drug Product Characteristics and Protocol Improve HbA T87Q Production • 51% HbA T87Q NEJM patient • Total Hb 12.6 g/dL 6 T 8 7 Q C o n c e n t r a t i o n ( g / d L ) Higher DP G r o u p A VCN G r o u p B 1 3 1 3 Higher in G r o u p B 1 3 1 2 vivo VCN 4 H G B - 2 0 5 Higher T87Q 2 H b A 0 0 1 2 3 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 N=9 patients 4 patients >6 months FU HbA T87Q 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
HbF production is controlled by a genetic ‘switch’ in stem cells 14
HbF Re-expression Strategies For Hemoglobinopathies Chr 2 Prevent Repressor expression (BCL11A enhancer disruption) x Interfere with BCL11a mRNA x Block Binding (recreate HPFH deletions) Chr 11
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 Advantages of Crispr-Cas9 TALENs HEs CRISPR/Cas9 (class) 1. High efficiency (in-del) ZFNs Mega-Tal 2. Precise DSB (less off-target) DNA Scissors 3. Conserves endogenous promoters and regulatory elements 4. Easy manufacturing/cost Homologous Non-homologous Recombination end-joining Donor DNA (copy and paste) (stitching) * Precise Spatial Modification Precise Spatial AND Nucleotide (DSB stimulates process by Modification of Genome >10 10 ) (DSB stimulates process by >10 5 )
Pros and Cons of gene-insertion versus gene-edit 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 Vector to provide promoters Uses endogenous regulation gene expression and regulatory elements Each Nuclease- Efficacy Transduction efficiency In-del efficiency, or HDR efficiency Variable 1. Extrapolation capability Safety Recombinant HIV no 2. Efficiency ‘off - target’ activity 3. Precision (‘off - target’) Insertional oncogenesis Cost High Low 18
Crispr Therapeutics Approach: Results of editing in healthy donor stem cells 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 Soni,S. pre-clinical development 19
Stanford Approach Cas9/gRNA (100 nt) complex (RNP) Homologous Recombination Donor DNA (AAV)
Editing E6V Sickle Mutation in Multiple SCD Patients 40 49% HR 30 % of Methyl Clones S/S 20 10 60 clones (3 patients) 0 HbS/HbS INDEL/HbS INDEL/INDEL HbA/HbS HbA/INDEL HbA/HbA
High Frequencies of Gene Correction at HBB in Patient Derived CD34+ HSPCs that is maintained after transplantation into NSG mice 80 70 60 50 % alleles 40 30 20 10 0 Sickle INDELs HR Allele Frequency in Human Allele Frequency in CD34+ Cells at 16 Weeks Post- HSPCs Prior to Transplant into Transplant into NSG Mice NSG Mice
Approaches to correct HSC for SCD Stanford UCSF Boston Children’s Crispr Tx St. Jude’s/ Editas bluebird bio Sangamo UCLA Univ.of Cincinnati TIGET, Milan Adapted from Blood, Feb,2016,127 23
Key Takeaways • 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 24
How do I choose which technology is better? 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 25
Questions
Rate Limiting Steps and Future 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 27
Antisickling Properties of β A-T87Q -Globin Chain Lateral Contact 87 b1 a1 Val 6 100 Phe 85 g -globin HLDDLKGTFA Q LSELHCDKLHVDPENF Leu 88 | | | : | | | | | | | | | | | | | | | | | | | | | | b -globin HLDNLKGTFA T LSELHCDKLHVDPENF b2 a2 | | | | | | | | | : | | | | | | | | | | | | | | | | d -globin HLDNLKGTFS Q LSELHCDKLHVDPENF b1 Val 6 a1 Phe 85 Leu 88 Axial Contact b2 F F a2 Q V V T b1 L Val 6 L Phe 85 a1 Leu 88 polymerization destabilization b2 a2 Confidential - 28
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