Promoterless Gene Targeting Approach Combined to CRISPR/Cas9 Efficiently Corrects Hemophilia B Phenotype in Neonatal Mice

Abstract

Many inborn errors of metabolism require life-long treatments and, in severe conditions involving the liver, organ transplantation remains the only curative treatment. Non-integrative AAV-mediated gene therapy has shown efficacy in adult patients. However, treatment in pediatric or juvenile settings, or in conditions associated with hepatocyte proliferation, may result in rapid loss of episomal viral DNA and thus therapeutic efficacy. Re-administration of the therapeutic vector later in time may not be possible due to the presence of anti-AAV neutralizing antibodies. We have previously shown the permanent rescue of the neonatal lethality of a Crigler-Najjar mouse model by applying an integrative gene-therapy based approach. Here, we targeted the human coagulation factor IX (hFIX) cDNA into a hemophilia B mouse model. Two AAV8 vectors were used: a promoterless vector with two arms of homology for the albumin locus, and a vector carrying the CRISPR/SaCas9 and the sgRNA. Treatment of neonatal P2 wild-type mice resulted in supraphysiological levels of hFIX being stable 10 months after dosing. A single injection of the AAV vectors into neonatal FIX KO mice also resulted in the stable expression of above-normal levels of hFIX, reaching up to 150% of the human levels. Mice subjected to tail clip analysis showed a clotting capacity comparable to wild-type animals, thus demonstrating the rescue of the disease phenotype. Immunohistological analysis revealed clusters of hFIX-positive hepatocytes. When we tested the approach in adult FIX KO mice, we detected hFIX in plasma by ELISA and in the liver by western blot. However, the hFIX levels were not sufficient to significantly ameliorate the bleeding phenotype upon tail clip assay. Experiments conducted using a AAV donor vectors containing the eGFP or the hFIX cDNAs showed a higher recombination rate in P2 mice compared to adult animals. With this study, we demonstrate an alternative gene targeting strategy exploiting the use of the CRISPR/SaCas9 platform that can be potentially applied in the treatment of pediatric patients suffering from hemophilia, also supporting its application to other liver monogenic diseases. For the treatment of adult patients, further studies for the improvement of targeting efficiency are still required.

In vivoexpansion of gene-targeted hepatocytes through transient inhibition of an essential gene

“…Similarly, in recent work the Alb-targeted expression of FIX efficiently corrected Hemophilia B in neonatal mice, yet it was inadequate in adult mice. 25 To increase the efficiency of gene targeting at the Alb locus, Tsuji et al administered ribonucleotide reductase inhibitors to neonatal and juvenile mice resulting in enhanced targeted integration. 8 However, the effectiveness of this strategy still needs to be evaluated in the adult liver, where HDR is rare.…”

Targeted Gene Insertion: The Cutting Edge of CRISPR Drug Development with Hemophilia as a Highlight

“…HDR accurately corrects or removes a DNA lesion through synthesizing new DNA based on existing homology sequences [ 28 ]. Targeted gene insertion via the HDR mechanism requires donors carrying flanking homology arms of approximately 0.6–1.4 kb [ 29 – 35 ]. The long homologies facilitate the HDR repair of not only site-specific DSBs induced by a nuclease but also endogenous DNA lesions within a large region, which can lead to low frequency gene insertion in the absence of nuclease.…”

Recent Advances in In Vivo Somatic Cell Gene Modification in Newborn Pups

“…Furthermore, neonatal hepatocytes exhibit active proliferation; however, proliferation ceases in the adult stage. Lisjak et al [121] suggested caution when F9-KO newborn mice are subjected to tail vein injection of rAAV carrying human F9 cDNA to rescue the bleeding phenotype. The treated mice had detectable levels of F9 in the plasma and liver.…”

“…This is due to the nature of juvenile hepatocytes, which show active cellular proliferation; during active cell proliferation, the rAAV vectors introduced inside a cell are lost. Based on these experiments, Lisjak et al [121] decided to use the CRISPR/SaCas9 platform, which could potentially be applied to the treatment of young patients with hemophilia. In contrast to juvenile hepatocytes, CNS neurons and skeletal muscle cells are non-proliferative.…”