Gene therapy based on recombinant adeno-associated virus (rAAV) vectors is a promising therapeutic approach. Recently, this strategy has shown promising treatment effects in clinical trials for various diseases, such as inherited retinal dystrophy (Russell et al., 2017), spinal muscular atrophy (Mendell et al., 2017), haemophilia (Nathwani et al., 2011; Rangarajan et al., 2017) and others (High and Roncarolo, 2019). However, preclinical studies have shown only short-term effects when the rAAV vector was delivered to target proliferating tissues in infant animal models (Cunningham et al., 2009; Wang et al., 2012). Moreover, a 3-year follow-up clinical trial for haemophilia A showed a continuous decline in FVIII levels over time in adult patients (Perrin et al., 2019), indicating that rAAV-mediated gene replacement therapy may be limited by the durability of treatment because rAAV vectors predominantly form episomes that are lost during cell proliferation (Davidoff and Nathwani, 2016).

The CRISPR/Cas9 system is a widely used genome editing technology that induces site-specific double-strand breaks (DSBs) with the guidance of single-guide RNA (sgRNA) (Li et al., 2022). DSBs can be repaired either through an error-prone nonhomologous end joining (NHEJ) pathway or a precise homology-directed repair (HDR) pathway in the presence of exogenous DNA templates (Rodgers and McVey, 2016). A CRISPR/Cas9-mediated HDR strategy has been successfully used to correct genetic disorders through correction of disease-causing genetic mutations or targeted integration of exogenous DNA at a target site, including haemophilia (Guan et al., 2016; Ohmori et al., 2017; Wang et al., 2019b), phenylketonuria (Yin et al., 2022) and ornithine transcarbamylase deficiency (OTCD) (Yang et al., 2016; Wang et al., 2020a). However, although HDR is highly accurate, its efficiency is limited even when delivered through the rAAV vector, whose single-stranded DNA genome is considered to induce a higher HDR rate (Gaj et al., 2016; Zhang et al., 2017). NHEJ is active throughout the cell cycle (Mao et al., 2008). The NHEJ-mediated targeted integration strategy provides an alternative approach to insert exogenous DNA in adults. In addition, this strategy is independent of the homologous arm to insert relatively larger DNA fragments, especially using an rAAV vector whose maximum capacity is approximately 4.7 kb ≤5 kb) (Wang et al., 2019a). Recently, a CRISPR/Cas9-mediated homology independent targeted integration (HITI) strategy was developed to efficiently target knock-in exogenous genes in dividing and nondividing cells. Importantly, through an innovative design of insertion of CRISPR/Cas9 recognition sequences within donor templates, the preferred orientation of inserted DNA could be determined with a high probability through HITI. With this strategy, retinitis pigmentosa retina was partially rescued in a rat model, suggesting that HITI is also functional in nondividing cells in vivo (Suzuki et al., 2016).

The albumin (Alb) locus is an attractive locus for gene integration that has high transcriptional activity (ALB comprises more than 25% of the synthetic proteins in the liver) and is considered a "safe haven" for integration. Several studies used the Alb locus as a targeted integration site, which successfully promoted the therapeutic effect compared with the disease locus itself in a variety of diseases (Li et al., 2011; Anguela et al., 2013; Barzel et al., 2015; Sharma et al., 2015; Conway et al., 2019; Wang et al., 2019b; Wang et al., 2020b; Chen et al., 2021). At present, a zinc finger nuclease (ZFN)-based targeted integration of the Alb locus for the treatment of mucopolysaccharidosis and haemophilia has been assayed in clinical trials (NCT02702115, NCT03041324, NCT02695160).

To investigate the potential of HITI to treat diseases in target organs other than the retina, we generated a haemophilia B (HB) model via CRISPR/Cas9 editing of the F9 gene in rats, since factor IX (FⅨ) is expressed mainly in hepatocytes, which are a critical cell type for gene therapy not only to treat diseases caused by hepatocyte dysfunction but also for the production of therapeutic secreting factors. HB is an ideal candidate to test novel strategies for gene therapy targeting hepatocytes, as our previous study demonstrated that moderate correction efficiency (>0.56%) would significantly ameliorate disease symptoms in mice (Guan et al., 2016). Moreover, FⅨ is a secreted protein that is a typical factor in enzyme replacement therapy. In this study, we tested the feasibility of the HITI strategy to treat HB through targeted insertion of high-specificity-activity Factor IX variant (F9 Padua, R338L) into intron 13 of the rat Alb (rAlb) gene locus. Through rAAV8-delivered Cas9/sgRNA and template in F9-deficient adult animals, the circulating FIX level was increased up to 52% of the normal level during 9 months of observation, demonstrating the amelioration of HB.