LENZ Therapeutics, Inc. · NASDAQ:LENZ
LENZ Therapeutics — Technical Presentation (June 2022)
16 slides from LENZ Therapeutics's technical presentation, June 2022
Slide 1: Development of a Beta-globin Gene Replacement Strategy for β-Thalassemia
Abstract #66 Development of a Beta-globin Gene Replacement Strategy as a Therapeutic Approach for β-Thalassemia Beeke Wienert, Kirby Wallace, Christopher Bandoro, Aishwarya Churi, James Partridge, Rajiv Sharma, William Matern, Sebastian Treusch, Daniel P. Dever Graphite Bio, Inc., South San Francisc
Slide 2: Disclosures
Disclosures All authors are current employees and equity holders in Graphite Bio. Christopher Bandoro reports equity holdings in Sana Biotechnology and James Partridge reports equity holdings in Global Blood Therapeutics. 2
Slide 3: Our goal: Restore normal genotype and physiology in people with hemoglobinopathies
Our goal: Restore normal genotype and physiology in people with hemoglobinopathies Sickle Cell Disease (SCD)1,2 β-Thalassemia2 Annually 330,000 infants are born with hemoglobinopathies worldwide, of which sickle cell disease and β-thalassemia are the most common3 1. Piel3FB, et al. Nat Commun. 2010;
Slide 4: The UltraHDR platform allows for gene correction and gene replacement
The UltraHDR™ platform allows for gene correction and gene replacement in sickle cell disease and β-thalassemia Dysfunctional β-globin gene Trial in Progress – Poster 806 High-fidelity (HiFi) CRISPR-associated protein 9 (Cas9) (Sequence-specific double-strand break) Donor Vector (adeno-associated vi
Slide 5: β-thalassemia is a genetic disorder with high unmet need
β-thalassemia is a genetic disorder with high unmet need characterized by reduced production of β-globin1 α β α Insoluble α-globin aggregates Beta-thalassemia major (β0) β α α Abnormal erythroblast Ineffective erythropoiesis No functional β-globin (most erythroblasts die in bone marrow) • More than
Slide 6: How do β-globin expression levels from a replaced coding sequence compare to physiological levels?
How do β-globin expression levels from a replaced β-globin coding sequence compare to physiological β-globin levels? Replaced β-globin gene Physiological β-globin expression HBB gRNA Diverged HBB exons (ex) HBB gRNA intron 1 intron 2 3’UTR β-globin intron 1 intron 2 3’UTR β-globin ex1 ex2 ex3 vs. ex
Slide 7: The β-globin gene requires introns for physiological protein expression
The β-globin gene requires introns for physiological protein expression Replaced β-globin gene Physiological β-globin expression HBB gRNA Diverged HBB exons HBB gRNA intron 1 intron 2 3’UTR β-globin intron 1 intron 2 3’UTR β-globin ex1 ex2 ex3 vs. ex1 ex2 ex3 E6V E6V β-globin ex1 ex2 ex3 pA Point mu
Slide 8: A T2A-EGFP reporter system allows screening for high-expressing DNA donors
A T2A-EGFP reporter system allows screening for high-expressing DNA donors Physiological β-globin control: HBB STOP gRNA Physiological expression ex1 intron 1 ex2 intron 2 ex3 3’UTR β-globin Cas9 only EGFP pA HBB-EGFP (ctrl) AAV6 only T2A β-globin gene replacement: HBB-EGFP (ctrl) HBB gRNA Diverged
Slide 9: Heterologous intron donors produce hemoglobin tetramers
Heterologous intron donors produce hemoglobin tetramers Single-Nucleotide No introns 60 Polymorphism (SNP) Donor 60 R2=0.3032 R2=0.9851 40 40 % HDR % HDR 20 20 SNP No introns 0 0 0 20 40 60 0 20 40 60 % hemoglobin tetramers % hemoglobin tetramers 60 HBG2 introns 60 HBD introns 60 Primate introns 40
Slide 10: Can the HBG2 β-globin DNA donor be further optimized?
Can the HBG2 β-globin DNA donor be further optimized? T2A ex1 ex2 ex3 EGFP bGH HBG2-WT introns int1 int2 Intron length: Polyadenylation (pA) sequences: • Shorter intron sequences, and • We assessed if different pA sequences thereby a shorter gene cassette, could increase protein expression may resul
Slide 11: Optimization of the HBG2 β-globin DNA donor identified an additional donor with truncated introns
Optimization of the HBG2 β-globin DNA donor identified an additional β-globin DNA donor with truncated introns T2A ex1 ex2 ex3 EGFP bGH HBG2-WT introns int1 int2 Truncated Introns • Most intron deletions resulted in a relative loss of expression 100 MFI (% of HBB-EGFP) • We identified one truncated
Slide 12: β-globin gene replacement is effective in both healthy and SCD patient HSPCs
β-globin gene replacement is effective in both healthy and SCD patient HSPCs HDR alleles in WT HSPCs HDR alleles in SCD HSPCs HiFi Cas9 gRNA 50.3 60 46.4 60 33.9 % HDR alleles % HDR alleles Electroporation 34.6 35.1 36.2 40 40 HDR alleles (digital droplet Gene-corrected polymerase WT/SCD 20 20 HSPCs
Slide 13: β-globin gene replacement restores adult hemoglobin (HbA) expression in patient SCD-HSPCs
β-globin gene replacement restores adult hemoglobin (HbA) expression in patient SCD-HSPCs SCD- HSPCs HbA/Sickle hemoglobin (HbS) (CD34+) RBC differentiation levels Gene-corrected (HPLC) SCD-HSPCs (CD34+) SCD-HSPC donors = 2 HPLC Untreated HBG2-WT Replicates = 6-8 8 10 100 % Globin tetramers HbA 8 Ab
Slide 14: Conclusions
Conclusions HiFi Cas9 gRNA • β-thalassemia is a genetic disorder with Electroporation high unmet need • Using our UltraHDR™ platform, we developed a precise β-globin gene HSPCs Gene-corrected replacement strategy that restores HbA (CD34+) HSPCs AAV6 DNA donor (CD34+) expression, offering a different
Slide 15: Acknowledgments
Acknowledgments • Graphite Bio team members: Jane Grogan, Shaheen Kabir, Vincent Siu, Glen Chew, Craig Ennis • Dr. John Tisdale, NIH, for providing SCD patient HSPCs Medical writing assistance was provided by Kymberleigh Romano, PhD, of Healthcare Consultancy Group, LLC and was 15 funded by Graphite
Slide 16: List of Abbreviations
List of Abbreviations AAV6, adeno-associated virus serotype 6 HBG2, hemoglobin subunit gamma 2 AU, absorbance unit HbS, sickle hemoglobin bGH, bovine growth hormone HDR, homology directed repair bp, base pair HiFi, high-fidelity Cas9, CRISPR-associated protein 9 HPLC, high-performance liquid chromat
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