Therapy Development by Genome Editing of Hematopoietic Stem Cells

Abstract

Accessibility of hematopoietic stem cells (HSCs) for the manipulation and repopulation of the blood and immune systems has placed them at the forefront of cell and gene therapy development. Recent advances in genome-editing tools, in particular for clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) and CRISPR/Cas-derived editing systems, have transformed the gene therapy landscape. Their versatility and the ability to edit genomic sequences and facilitate gene disruption, correction or insertion, have broadened the spectrum of potential gene therapy targets and accelerated the development of potential curative therapies for many rare diseases treatable by transplantation or modification of HSCs. Ongoing developments seek to address efficiency and precision of HSC modification, tolerability of treatment and the distribution and affordability of corresponding therapies. Here, we give an overview of recent progress in the field of HSC genome editing as treatment for inherited disorders and summarize the most significant findings from corresponding preclinical and clinical studies. With emphasis on HSC-based therapies, we also discuss technical hurdles that need to be overcome en route to clinical translation of genome editing and indicate advances that may facilitate routine application beyond the most common disorders.

Keywords: CRISPR/Cas; TALEN; ZFN; base editor; blood disorders; gene therapy (GT); genome editing; hematopoietic stem cell; monogenic disorder; prime editor.

Figure 1

Overview of exemplary hereditary disorders potentially curable by editing of HSCs. Genes associated with each disease are indicated in parentheses. The cell types given indicate where protein expression or phenotypic correction are most apparent. Note that combined immunodeficiencies affect B cell function even when presenting with a B⁺ phenotype. Monocytes and macrophages (MΦ) may also act on cells and for disease correction outside the hematopoietic system (not shown). RBC—red blood cell, NK cell—natural killer cell.

Figure 2

Structure, function and HSC-based application of common gene-editing platforms. ZFNs, TALENs and CRISPR/Cas9 are chimeric proteins comprising customizable sequence-specific DNA binding domain (e.g., zinc finger—ZF, transcription activator-like effector proteins—TALEs or single guide RNA—sgRNA) and a nonspecific nuclease that mediates DNA cleavage (e.g., FokI nuclease in the context of ZFNs and TALENs and Cas9 in the case of CRISPR/Cas9). DNA double-strand breaks generated by ZFNs, TALENs and CRISPR/Cas9 are mainly repaired via two endogenous pathways: (1) error-prone non-homologous end joining (NHEJ), which occurs throughout the cell cycle and corrects breaks through ligation of DNA ends, or (2) by precise homology-directed repair (HDR), in the presence of donor template provided, e.g., as synthetic single-stranded oligodeoxynucleotides (ssODNs), insertion-defective lentiviral vector (IDLV) or adeno-associated virus 6 vector (AAV6) components,. Base editors are chimeric proteins composed of a mutated nuclease, such as Cas9 nickase (nCas9), a catalytic domain capable of deaminating a cytidine or adenine base to induce transition mutations, and a uracil glycosylase inhibitor to prevent base excision repair of the transition event. Prime editors are chimeric proteins exploiting an extended gRNA, termed prime editing guide RNA (pegRNA), and a nCas9 fused to a reverse transcriptase, which nick the DNA to allow pegRNA binding of flanking gRNA to serve as primer of pegRNA-directed reverse transcription of the desired sequence change.

Figure 3

Ex vivo vs. in vivo HSC gene-editing. Steps shown for Ex vivo editing are widely applied for gene editing in in vivo animal models and now also in clinical trials for gene editing. Findings for therapy by gene addition indicate that gene editing, too, might benefit from selective HSC depletion by delivery of antibody-drug conjugates [55] and for suitable disorders, such as FA, from engraftment of corrected cells without conditioning [14]. Steps shown for In vivo editing are in part extrapolated for HSC-targeted approaches of gene addition [56,57,58] and in part based on the latest developments and concepts in the delivery of gene editing components and mRNAs [59,60,61,62].