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CRISPR gene editing to cure Sickle cell disease : Hurdles to overcome in Stem Cell Transplantation developing an IRB approved for Sickle Cell Disease clinical trial Fred Goldman MD Director, Pediatric Blood and Marrow Transplant Program Children’s of Alabama, University of Alabama, Birmingham Outline Review sickle cell disease and therapies Review bone marrow transplant and gene therapy Discuss CRISPR technology Show our preclinical data in mice and humans Safety concerns and FDA approval How to best protect human subjects Sickle cell disease (SCD) Most common genetic disorder amongst African Americans Caused by a single base pair mutation in the betahemoglobin chain GAG to GTG at codon 6 Glutamic Acid to Valine substitution produces hydrophobic projection Abnormal hemoglobin polymerizes upon deoxygenation, RBC to take on its characteristic sickle shape Consequence is severely shortened red blood cell lifespan and severe anemia Epidemiology and statistics The average life expectancy: 42 years for males and 48 years for females Approximately 1 in 600 African American births results in sickle cell disease About 100,000 Americans have sickle cell disease, 2000 new births annually 1000 children with SCD in Alabama, ~100 between 18-21, ~8,000 adults Newborn screening 50 new cases/yr in Alabama Nigeria, 150,000 born with SCD annually This is a WORLD WIDE problem Long term organ damage in SCD Neurologic Pulmonary issues Acute chest syndrome Pulmonary hypertension 40% by age 40 Bone infarctions 11% will have a stroke before age 18 1/3 will develop silent stroke by age 15 Transcranial doppler (TCD) predictive Cognitive dysfunction Renal insufficiency Infection risks Vasocclusive crises = pain crises Iron overload from chronic transfusion also causes end organ damage Current treatment options in SCD Supportive care-manage symptoms Blood transfusions for acute event Hydroxyurea (HU) Increases Hgb F, lowers white blood cell count Clinical trials Prophylactic antibiotics pain medications SIT trial- transfusion better than observation STOP trial- need transfusion for abnormal transcranial doppler study SWITCH- transfusion better than HU in preventing strokes Baby HUG- fewer crises with HU vs placebo Hematopoietic Stem Cell Transplantation Allogeneic hematopoietic stem cell transplantation Bone marrow harvested from normal donor Patient with disease Chemotherapy to wipe out disease and create space Stem cell infusion Medication to prevent graftversus-host disease (GvHD) Source of hematopoietic stem cells for transplant Bone Marrow Peripheral blood stem cells CD34+ Cord Blood BMT for sickle cell disease Only curative option Donor identification is a problem 15% will have matched-sibling donor 60% will have HLA-matched unrelated donor (MUD) >25% have no suitable MUD Due to toxicities, BMT is reserved for those with severe disease Recurrent chest syndrome CNS event Recurrent severe pain episodes Impaired neuropsychological functioning with abnormal imaging Transplant outcomes by donor type Matched related donor Other donor types Expected complications of allogeneic BMT Chemotherapy mediated Graft Failure Primary rejection Secondary rejection-occurs after 2 months Acute or chronic, higher with MUD Neurologic*** increased incidence in SCD Hepatic Cardiac Hematologic toxicity, bleeding Increase infection risk from low white count Mucositis, pain Graft versus Host Disease Other organ specific complications Reduced intensity regimens for SCD and mixed donor chimerism Less chemo=less toxicity in graft rejection Partially engrafted in WBC, yet fully engraft with RBC Hsieh et al, NEJM, 2009 Non-SCD Donor Sickle Trait Donor Hgb S Donor chimerism Hgb S Donor Chimerism 0 67 36 25 0 74 37 60 7 11 Wu et al, BJH, 2007 Conclusion – “mixed chimerism is a suitable endpoint of stem cell-based therapies for SCD” Gene therapy • Introduction of new genetic material into the cells of an organism for therapeutic purposes • Abnormal gene and normal functioning gene identified and cloned • Cells responsible for disease are identified and accessible for manipulation • A means of introducing and expressing genetic material (viral vector/gene editing) • Over 2300 clinical trials from 1989-2016 (per Wikipedia) Gene therapy for disorders of the hematopoietic system Severe immune deficiencies Common g chain ~50 pts ADA deficiency ~20 pts CGD ~ 5 pts WAS ~ 15 pts Marrow failure disorders Fanconi’s anemia 7 pts Hemoglobinopathies Sickle cell disease ~ 5 pts Thalassemia ~15 pts Steps in ex-vivo gene therapy Sustained Correction of X-Linked Severe Combined Immunodeficiency by ex Vivo Gene Therapy • • • • 12 males with X-linked SCIDS Bone marrow derived CD34+ HSC Ex vivo expanded transduced using retroviral vector containing common g chain Hacein-Bey-Abina et al, NEJM 2002 Gene therapy complication insertional mutagenesis LMO2-Associated Clonal T Cell Proliferation in Two Patients after Gene Therapy for SCID-X1 Hacein-Bey-Abina et al, Science 2003 BlueBird Bio Phase 1 study HGB-206 for SCD: Gene therapy using autologous CD34+ cells transduced ex vivo with lentiglobin BB305 lentiviral vector Eligibility 18 or older Severe SCD Primary endpoint: safety Success and kinetics of HSC engraftment Incidence of mortality Detection of vector mediated insertional mutagenesis Clinical adverse events Data from Bluebird study reported Bone marrow harvesting Transduction efficiency of HgbAT87Q Transfused to Hgb 10-12 with <30% HgbS Harvest volume 15-20 ml/kg, repeated to obtain minimum .5-1.3 vector copy number/CD34+ cell pre infusion .1 vector copy/peripheral blood leukocyte 2-3 months post Full dose myeloablation Busulfex 3.2 mg/kg IV x 16 dose, AUC=1100 Hematologic toxicity expected Neutrophil engraftment d16-18 Platelet engraftment d 23-29 Outcomes Patients transfusion dependent with minimal expression of HgbAT87Q Safety summary Bone marrow harvest 2 non serious grade 3 SAE- pain 1 serious SAE with prolonged hospitalization due to pain N=4, 2,2,2 and 4 harvests needed to achieve volume 1.9-3.2L TNC 7-12 x 10>8/kg Safety of infused subjects No AE from BB305 infusion 1 serious AE d+42 (bacteremia) 2 serious AE >d+42 pain crisis 9 non-hematologic grade 3-4 AE in 2 subjects Fever, mouth pain, mucositis, febrile neutropenia, anorexia, fatigue, dyspnea (gr3), bacteremia (gr 4) Safety profile consistent with autologous HSCT Problems with current gene addition therapy trials Failure to control integration sites Random gene insertion in or near proto oncogenes Limited cell numbers Common gamma chain SCIDs 5/20 WAS trial 5/10 CGD 5/5 due to primary disease state Age of patient and challenge of harvest Inefficiency/inconsistency of transduction Not under control of endogenous regulators CRISPR-Cas System (Clustered Regularly Interspersed Palindromic Repeats) CRISPR-Cas System Provides bacteria with innate immunity to defend against invading viruses Gene correction via CRISPR/Cas • The CRISPR/Cas system has been used for gene editing (adding, disrupting or changing the sequence of specific genes) • By delivering the Cas9 protein/enzyme and appropriate guide RNAs into a cell, the genome can be cut/repaired at any desired location Phase I Trial of PD-1 Knockout Engineered T Cells for the Treatment of Metastatic Nonsmall Cell Lung Cancer Evaluate the safety of CRISPR-mediated PD-1 knockout engineered T cells Primary outcomes Number of participants with adverse events and/or dose limiting toxicities as a measure of safety Sponsor Sichuan University UAB IRB approved protocol to collect bone marrow from SCD patients Sickle cell adult patients consent to undergo a bone marrow aspirate Patients financially compensated Collect 30 ml of marrow from hip Isolate CD34 cells by selection column Test in vitro and in NSG mice Differentiation in vitro of CRISPR/Cas corrected sickle CD34+ stem cells CRISPR/Cas modified to allow optimal correction and preserve viability CD34+ cells undergo nucleoporation with RNP/ssODN complex Cells are plated onto methocult where they differentiate Red blood cell precursor colonies are picked and DNA is sequenced CD34+ BFU-E GEMM S/S Selected Sanger sequencing results of six genotypes of GEMM/BFU-E colonies S/Indel Indel/Indel A/S A/Indel A/A mr5Cas9 complex nucleofected human sickle CD34+ GEMM/BFU-E sanger sequencing results 1 2 3 4 5 6 7 8 9 10 11 12 A SS A/IND A/S SS SS A/IND SS SS A/S IND/IND A/S S/IND B SS A/S SS S/IND S/IND SS SS AA S/IND SS AA SS C SS A/IND SS SS IND/IND A/S S/IND SS S/IND SS A/S SS D S/IND SS S/IND AA SS SS IND/IND SS SS A/S SS A/IND E SS SS S/IND SS A/S SS A/S S/IND S/IND SS SS IND/IND F IND/IND SS SS A/IND SS SS A/IND IND/IND SS SS A/S SS G IND/IND SS SS A/S IND/IND A/IND A/S A/IND SS A/S S/IND A/IND H SS SS S/IND SS SS S/IND SS S/IND A/S SS SS A/IND Complex for nucleofection mr1Cas9 + T2gRNA + ssODN GEMM /BFU-E colonies Total colonies A/A A/S S/S A/indel S/indel Indel/indel Colonies with at least 1 allele corrected Colonies with indels Colonies with genome modification Total number of alleles 16/80 96 3.1% 14.6% 47.9% 10.4% 15.6% 8.3% 28.1% 42.7% 52.1% 3 14 46 10 15 8 27 41 50 192 Total “A” alleles (corrected) 30 Total “S” alleles (uncorrected) Total “indel” alleles 121 41 15.6% 63.7% 21.6% GEMM correction: 4/16 Total Colonies: 2.5X CAS9WT Deep sequencing data for 5 Off-Target (OT) sites in Sickle BM CD34+ GEMM/ BFU-E 0.1200% 0.1000% 0.0800% Neg ctrl wtCas9 0.0600% mr5Cas9 mr4Cas9 0.0400% 0.0200% 0.0000% OT1 OT2 OT3 OT4 OT5 CRISPR corrected human sickle CD34+ bone marrow produce RBCs with normal hemoglobin A Qualitative mass spec analysis of betaglobin in sample marked with red rectangle above. Quantitative data obtained from comparison to a standard curve of known mixtures of betaS and betaA peptides demonstrate 35% betaA. Mouse model of sickle cell disease Science 245: 971-973 (1989) Science 247: 566-568 (1990) Science 278: 873-876 (1997) Science 318:1920-1923 (2007) Sickle gene correction in long-term reconstituting HSC mouse control mouse AS control mouse AA control mouse SS control mr5Cas9-BM#30 mr5Cas9-BM#10 mr5Cas9-BM#3 mr5Cas9-BM#1 IEF gel analysis of hemoglobin at 12 weeks after secondary transplant (24 weeks total) Blood counts 12 weeks after secondary transplant RBC (x10^6 cells/µL) HGB (g/dL) HCT (%) % Retic AS SS Corrected 11.26 10.4 46.1 17.47 6.38 6.5 34.4 56.8 10.53 9.4 40.5 6.38 Spleens at 12 weeks after secondary transplant AA Control SS Corrected SS Uncorrected control Human A/S (sickle trait) cord blood CD34+ mr5Cas9/ssODN nucleoporation Intra-femoral injection into NSG mice FACS Purification of BM Cells at 13 Weeks Post-Transplantation Multi-lineage long-term reconstitution of corrected human CD34+ cells in NSG Mice FACS Sorted Cells A*/A*+ S B cells (CD19) T cells (CD7) monocyte RBC pre Stem cell (CD11b) (CD235a) (CD34) Exp 1 Exp 2 2.00E+05 2.60E+05 1.10E+05 1.80E+05 3.30E+05 3.20E+05 1.80E+04 4.10E+04 2.60E+05 7.80E+05 Exp 1 Exp 2 6.00% 60.70% 6.80% 78.10% 7.70% 82.20% 5.40% 66.70% 2.80% 72.20% Summary of pre-clinical data CRISPR/Cas complex can correct the sickle mutation in both human and murine HSC Efficiency of correction (25-50%) should be sufficient to correct the disease CRISPR can correct true HSC as demonstrated by secondary transplants Minimal off target sites and no evidence of mutagenesis thus far Challenges for a phase 1 study Collection of HSC from sickle cell patients Preparative therapy Must complete preclinical safety studies in mice transplanted with “therapeutic dose” Patient enrollment UAB GMP facility Safety issues of marrow collection Requires sufficient volume to get desired CD34 cell count General anesthesia and transfusions Need 2-3 liters of marrow Recovery and pain post procedure, repeat? Alternatives GCSF mobilization of peripheral blood stem cells not feasible in SCD Expansion techniques ex vivo, successful for cord blood (SR-1) Safety issue of chemo-ablation Full dose Busulfan and associated toxicity Partial dose Busulfan (25%) Works for SCIDs In sickle cell disease, may be inadequate for engraftment Alternatives Non myeloablative conditioning and use of marrow niche clearing agents (ACK2) Preclinical testing ongoing in sickle mice Successful in other murine disease models CRISPR issues Must produce in bulk in a GMP facility Consistent nucleoporation and correction efficiency Malignant transformation potential low Monitor sites and sequence off target and oncogene Antibody formation Patient issues Who would be the best candidate What side effects will be expected What if there is no engraftment What the FDA will say? Medical standards are disease specific CRISPR editing of sickle CD34+ cells is more than “minimal manipulation” and thus will be considered a “drug” therapy needing an IND GMP facility for stem cell processing and CRISPR production Work with local IRB in tandem in developing plan Comparison of transplant options Allogeneic MUD HSCT Allogeneic Haplo HSCT Allogeneic MRD HSCT Lentivi HSCT CRISPR HSCT donor 60% 100% 15% 100% 100% GVHD +++ +/++ + - - Infection ++ ++ ++ + + reject + + + ++ ? # done ~100 ~100 ~500 5 0 Cost ~ 600k ~ 600k ~ 400k ? ? Acknowledgements Tim Townes and the lab Lei Ding Chao Li Chai Wei Chang Li-Shin Lai Kevin Pawlik Joe Sun Jane Wu Dewang Zhou Erik Westin Divya Devadasan IRB UAB Hematology service OB/GYN Stem Cell Institute All of our sickle cell patients and their families