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Promoterless gene targeting without nucleases ameliorates haemophilia B in mice

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Site-specific gene addition can allow stable transgene expression for gene therapy. When possible, this is preferred over the use of promiscuously integrating vectors, which are sometimes associated with clonal expansion1 and oncogenesis2. Site-specific endonucleases that can induce high rates of targeted genome editing are finding increasing applications in biological discovery and gene therapy3. However, two safety concerns persist: endonuclease-associated adverse effects, both on-target4 and off-target5,6; and oncogene activation caused by promoter integration, even without nucleases7. Here we perform recombinant adeno-associated virus (rAAV)-mediated promoterless gene targeting without nucleases and demonstrate amelioration of the bleeding diathesis in haemophilia B mice. In particular, we target a promoterless human coagulation factor IX (F9) gene to the liver-expressed mouse albumin (Alb) locus. F9 is targeted, along with a preceding 2A-peptide coding sequence, to be integrated just upstream to the Alb stop codon. While F9 is fused to Alb at the DNA and RNA levels, two separate proteins are synthesized by way of ribosomal skipping. Thus, F9 expression is linked to robust hepatic albumin expression without disrupting it. We injected an AAV8-F9 vector into neonatal and adult mice and achieved on-target integration into 0.5% of the albumin alleles in hepatocytes. We established that F9 was produced only from on-target integration, and ribosomal skipping was highly efficient. Stable F9 plasma levels at 7–20% of normal were obtained, and treated F9-deficient mice had normal coagulation times. In conclusion, transgene integration as a 2A-fusion to a highly expressed endogenous gene may obviate the requirement for nucleases and/or vector-borne promoters. This method may allow for safe and efficacious gene targeting in both infants and adults by greatly diminishing off-target effects while still providing therapeutic levels of expression from integration.

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Figure 1: Vector design and experimental scheme.
Figure 2: Human F9 expression and activity in injected mice.
Figure 3: Rate of Alb targeting at the DNA and RNA levels.
Figure 4: Specificity of F9 expression.

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  • 04 November 2014

    Fig. 2e was corrected in the PDF owing to a production error.


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This work was supported by a grant to M.A.K. from the National Heart Lung & Blood Institute (R01-HL064274). A.B. was supported by a fellowship from the Lucile Packard Foundation for Children’s Health, Stanford NIH-NCATS-CTSA UL1 TR001085 and Child Health Research Institute of Stanford University. N.K.P. was supported by a fellowship from the National Heart Lung & Blood Institute (F32-HL119059), the Hans Popper Memorial Fellowship from the American Liver Foundation, and the Stanford Dean’s Fellowship. M.H.P. was supported by the Laurie Krauss Lacob Faculty Scholar Fund in Pediatric Translational Medicine. The funding organizations played no role in experimental design, data analysis or manuscript preparation.

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Authors and Affiliations



A.B., N.K.P., M.H.P., K.M.G. and M.A.K. designed the experiments. A.B., N.K.P., Y.S., Y.H., K.C., F.Z., P.N.V. and L.P.S. generated reagents and performed the experiments. A.B., N.K.P. and M.A.K. wrote and edited the manuscript.

Corresponding author

Correspondence to M. A. Kay.

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Competing interests

A.B. and M.A.K. are founders of LogicBio Therapeutics, a startup biotechnology company with interests in the technology described in the manuscript.

Extended data figures and tables

Extended Data Figure 1 Human F9 liver immunohistochemistry.

From top to bottom, panels show human F9 staining (red) with 4′,6-diamidino-2-phenylindole (DAPI) nuclear counterstain (blue) in positive control human liver, negative control untreated mouse liver, and two sets of representative stains from mice treated as neonates or adults with AAV8-F9. Original magnification, ×200.

Extended Data Figure 2 Scheme of targeting rate assessment.

Assessment of on-target integration rate begins using linear amplification with biotinylated primer 1 (black), annealing to the genomic locus but not to the vector (step 1). Linear amplicons are then bound to streptavidinylated beads and washed to exclude episomal vectors (step 2). Subsequent second-strand DNA synthesis with random primers (step 3) was followed by CviQI restriction digestion (step 4). A compatible linker is then ligated (step 5) followed by two rounds of nested PCR amplifications (primers 2–3 in blue (step 6), and then primers 4–5 in red (step 7)). CviQI cleaves at the same distance from the homology border in both targeted and wild-type alleles, thus allowing for unbiased amplification. The amplicons of the second nested PCR then serve as a template for qPCR assays with either primers 6–7 (green) or 8–9 (orange) (step 8).

Extended Data Figure 3 Standard curves for targeting rate assessment by qPCR.

qPCR standard curves for the targeted allele (primers 8 and 9, Fig. 3) and non-targeted allele (primers 6 and 7, Fig. 3). Mass units used are functionally equivalent to molarity because all amplicons used were of equal length.

Extended Data Figure 4 Toxicity assessment by ALT measurement.

Alanine transaminase levels (ALT) were evaluated 7 days after injection in mice injected with AAV8 coding for our experimental vector (1 × 1012) or a negative control coding for a known non-toxic cassette (1 × 1012 of H1 promoter-driven shRNA), or a positive control coding for a known toxic cassette (5 × 1011 of U6 promoter-driven shRNA). Data represent mean of two measurements of four independent mice for each groups. The statistical significance is defined here as having P < 0.05 in a one-tailed t-test between samples of different variance.

Extended Data Figure 5 Vector copy number.

Vector copy number assessed by qPCR using primers 8 and 9 (Fig. 3). n = 7 for mice injected as adults; n = 6 for mice injected as neonates and analysed before or after partial hepatectomy (PH). Error bars represent s.d.

Extended Data Table 1 Haplotypes in the human population at the relevant ALB locus as extracted from the 1000 Genomes Project

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Barzel, A., Paulk, N., Shi, Y. et al. Promoterless gene targeting without nucleases ameliorates haemophilia B in mice. Nature 517, 360–364 (2015).

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