Gene Therapy for Inherited Blood Disorders

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

Purpose and Scope The objective for this fiscal year was to advance an in vivo adenine base editing (ABE) platform for Fanconi anemia (FA) by addressing major delivery bottlenecks. Studies employed primary human CD34⁺ hematopoietic stem and progenitor cells (HSPCs), FA patient–derived dermal fibroblasts, Epstein–Barr virus (EBV)–transformed lymphoblastoid cell lines (LCLs), and rhesus macaques for pharmacokinetic and biodistribution analyses. The program leverages biospecimens and clinical insights from the principal investigator’s ongoing NIH eltrombopag trial in FA (NCT03206086). Experimental Methods Key methodologies included CRISPR-derived ABE systems; lipid nanoparticle (LNP) formulation and bioconjugation; flow cytometry; dynamic light scattering and zeta potential measurements; RiboGreen mRNA encapsulation assays; confocal microscopy; high-throughput and Illumina next-generation sequencing (NGS); sodium–iodide symporter (NIS) reporter positron emission tomography–computed tomography (PET-CT); and single-cell RNA sequencing (scRNA-seq). Delivery Engineering A high-affinity bispecific single-chain variable fragment minibody targeting the thrombopoietin receptor (biscFv[cMPL]) was generated. Conjugation using inverse electron-demand Diels–Alder (IEDDA) tetrazine–trans-cyclooctene (TCO) micelle insertion preserved LNP size, dispersity, payload retention, and ligand affinity. Using the novel dendrimer ionizable lipid 4A3-SC8, 63 LNP variants were produced; the lead formulation achieved >80% green fluorescent protein (GFP) expression in primary human HSPCs, outperforming standard comparators. In rhesus macaques, NIS PET-CT revealed predominant hepatic and splenic uptake of non-targeted LNPs. Single-cell RNA-seq confirmed macrophage-dominant uptake in liver, spleen, and marrow, supporting the development of CD47-peptide cloaking to attenuate mononuclear phagocyte system (MPS) clearance. Editor Selection and Locus Targeting Efforts focused on the recurrent FANCA c.2638C>T (p.Arg880*) mutation. In primary human HSPCs, electroporation of ABE8e variants with guide RNAs achieved >80% editing at a proximal FANCA site (c.2677), whereas unmodified LNPs yielded <4% editing, highlighting delivery as the major barrier addressed in Aims 1–4. In FA patient–derived fibroblasts and EBV LCLs harboring the R880* mutation, ABE8e-HiFi with guide 3 achieved ~12% and ~25% allelic correction, respectively, supporting selection of the ABE8e-HiFi/guide 3 combination for continued development. Data Resources Generated This year’s datasets include: • LNP formulation–performance matrices. • Physicochemical characterization (dynamic light scattering, zeta potential, mRNA encapsulation). • Flow cytometry–based uptake in CD34⁺ HSPCs. • NIS PET-CT biodistribution profiles. • Single-cell atlases mapping nanoparticle uptake. • Targeted NGS of FANCA for quantification of editing outcomes. These resources inform iterative optimization of c-MPL–targeted LNPs, CD47 cloaking, fusogenic peptide density, and upstream open reading frame (uORF)–enabled translation in quiescent HSPCs. Limitations and Risks Key barriers include: • Hepatic sequestration via apolipoprotein E/LDL receptor pathways. • MPS-mediated clearance. • Inefficient endosomal escape of large ABE transcripts. • Integrated stress response (ISR)–driven translational attenuation. Future Directions The next phase will integrate bone marrow targeting, MPS evasion, endosomal escape, and translational enhancement into a unified LNP platform. Planned evaluations in rhesus macaques will assess safety, biodistribution, editing efficiency, and hematopoietic function. Target benchmarks include: • Improved marrow uptake with reduced hepatic accumulation. • HSPC editing ≥5% sustained for ≥6 months. • Off-target editing <0.1%. • Restoration of function in FANCD2 and mitomycin C resistance assays.