Homology-Directed-Repair-Based Genome Editing in HSPCs for the Treatment of Inborn Errors of Immunity and Blood Disorders

Abstract

Genome engineering via targeted nucleases, specifically CRISPR-Cas9, has revolutionized the field of gene therapy research, providing a potential treatment for diseases of the blood and immune system. While numerous genome editing techniques have been used, CRISPR-Cas9 homology-directed repair (HDR)-mediated editing represents a promising method for the site-specific insertion of large transgenes for gene knock-in or gene correction. Alternative methods, such as lentiviral/gammaretroviral gene addition, gene knock-out via non-homologous end joining (NHEJ)-mediated editing, and base or prime editing, have shown great promise for clinical applications, yet all possess significant drawbacks when applied in the treatment of patients suffering from inborn errors of immunity or blood system disorders. This review aims to highlight the transformational benefits of HDR-mediated gene therapy and possible solutions for the existing problems holding the methodology back. Together, we aim to help bring HDR-based gene therapy in CD34⁺ hematopoietic stem progenitor cells (HSPCs) from the lab bench to the bedside.

Keywords: CRISPR-Cas9; HDR; gene editing; gene therapy.

Figure 1

Genome-editing Nucleases. (A) Schematic depicting ZFN and TALEN genome-editing systems and the resulting DSB. (B) Schematic depicting the CRISPR-Cas9 genome-editing systems and the resulting DSB.

Figure 2

Delivery Mechanisms for HDR. (A) Single-stranded oligodeoxynucleotides (ssODNs). Together with ssODNS, DSBs are processed by the RAD51-independent SSTR pathway, in which the ssDNA filament invades the homologous region to form a D-loop structure, enabling DNA synthesis by a polymerase and eventual resection of the DSB [66]. (B) ssDNA with dsDNA Cas9 target sequences on the ends. These donors have been shown to induce efficient HDR at donor lengths of up to ~3.5 kb. The NLS on the Cas9 allows for more efficient trafficking of the donor into the nucleus, enabling enhanced HDR efficiencies [67]. (C) Adenovirus 5/35 (AdV). AdVs are capable of carrying very large dsDNA payloads of ~30–35 kb. The 5′ termini of AdV genome bind covalently to virus-coded terminal proteins, enabling better stability and markedly reduced HDR-independent incorporation into the genome [68]. (D) Recombinant adeno-associated virus serotype 6 (AAV). AAV vectors carry an ssDNA payload with inverted terminal repeats that create dsDNA loops at both the 5′ and 3′ ends of the genome. After entering the cell, the genome is believed to undergo second-strand synthesis, providing a dsDNA intermediate to facilitate HDR and integration into the genome [69,70,71]. (E) Integration-deficient Lentivirus (IDLV). IDLVs carry single-stranded viral RNA along with other enzymes including reverse transcriptase. When the ssRNA payload is released into the cytoplasm it undergoes reverse transcription, producing a dsDNA product that can then be imported into the nucleus to serve as an HDR template [72].

Figure 3

HDR formulations: Insertion vs. Replacement. Both techniques utilize homology arms; however, whereas the insertion method (gray dotted lines) has homology arms centered at the Cas9 cut site (depicted as the red line downstream to the gene’s ATG), the replacement method (green dotted lines) has a left homology arm flanking the genomic DNA upstream to the cut site while the right homology arm is distanced to be downstream to the region being replaced, in the depicted case, downstream to the stop the codon of the gene’s open reading frame (ORF).