A comparison of DNA repair pathways to achieve a site-specific gene modification of the Bruton's tyrosine kinase gene

David H Gray,Jasmine Santos,Alexandra Grace Keir,Isaac Villegas,Simon Maddock,Edward C Trope,Joseph D Long,Caroline Y Kuo

doi:10.1016/j.omtn.2021.12.014

David H Gray, Jasmine Santos + Show 6 more

Open Access

https://doi.org/10.1016/j.omtn.2021.12.014

Copy DOI

Abstract

Gene editing utilizing homology-directed repair has advanced significantly for many monogenic diseases of the hematopoietic system in recent years but has also been hindered by decreases between in vitro and in vivo gene integration rates. Homology-directed repair occurs primarily in the S/G2 phases of the cell cycle, whereas long-term engrafting hematopoietic stem cells are typically quiescent. Alternative methods for a targeted integration have been proposed including homology-independent targeted integration and precise integration into target chromosome, which utilize non-homologous end joining and microhomology-mediated end joining, respectively. Non-homologous end joining occurs throughout the cell cycle, while microhomology-mediated end joining occurs predominantly in the S phase. We compared these pathways for the integration of a corrective DNA cassette at the Bruton’s tyrosine kinase gene for the treatment of X-linked agammaglobulinemia. Homology-directed repair generated the most integration in K562 cells; however, synchronizing cells into G1 resulted in the highest integration rates with homology-independent targeted integration. Only homology-directed repair produced seamless junctions, making it optimal for targets where insertions and deletions are impermissible. Bulk CD34+ cells were best edited by homology-directed repair and precise integration into the target chromosome, while sorted hematopoietic stem cells contained similar integration rates using all corrective donors.

Highlights

The identification of targeted endonucleases, the RNAguided clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins, has brought genome editing to the forefront in the field of gene therapy.[1,2] These endonucleases can create double-stranded breaks (DSBs) in genomic DNA with high efficiency and specificity, initiating one of several cellular repair pathways
Corrective Bruton’s tyrosine kinase (BTK) cDNA cassettes were designed to integrate at intron 1 because intronic regions are permissive to imperfect junctions that are expected to result from homology-independent targeted integration (HITI)- and precise integration into target chromosome (PITCh)-mediated integrations (Figures 1A and S1)
Patients with X-linked agammaglobulinemia (XLA) have loss-of-function mutations in the Bruton’s tyrosine kinase (BTK) gene, which encodes a cytoplasmic signaling protein found in most hematopoietic lineages.[22]

Summary

Introduction

The identification of targeted endonucleases, the RNAguided clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins, has brought genome editing to the forefront in the field of gene therapy.[1,2] These endonucleases can create double-stranded breaks (DSBs) in genomic DNA with high efficiency and specificity, initiating one of several cellular repair pathways. The most thoroughly described DSB repair pathways are non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ is an error-prone process that repairs the DSB while introducing random nucleotide insertions and deletions (indels) at the repair junction.[3] In contrast, HDR uses a homologous template, typically a sister chromatid during cell replication, for a seamless repair of the break.[4] Each of these two methods has been harnessed for different purposes. NHEJ is most commonly used to disrupt elements in the genome through the introduction of indels at the DSB, while HDR is used for the targeted correction of single base pairs (bp) or the insertion of large sequences of DNA for site-specific gene replacement.[5,6,7,8,9]

Methods

Results

Conclusion