Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo

In the paper

“The mean particle diameter of nano.Cas9 is about 120 nm as determined by dynamic light scattering (Supplementary Fig. 1c).”

In the paper

“Compared to indels at the on-target Fah site (Fig. 3f), <0.3% indels were detected at the assayed off-target sites in nano.Cas9 + AAV-HDR–treated mice, and these numbers were comparable with AAV-HDR–treated control mice (Supplementary Fig. 7 and Supplementary Table 6), indicating that Cas9 mRNA delivery has minimal off-target effects at assayed sites for Fah sgRNA.”

In the paper

“OT1 and several other assayed sites had modest indel rates, and none of these sites were substantially higher than the background indel rate in untreated livers (P > 0.05; Supplementary Fig. 8b and Supplementary Table 9).”

In the paper

“We observed ∼24% indels and ∼1.2% beta-Catenin–positive hepatocytes in vivo (Supplementary Fig. 6).”

In the paper

“We observed a clear band in livers of nano.Cas9 + AAV-HDR–treated animals, but not in control animals (Supplementary Fig. 5b). We sequenced the band and identified that the corrected sequence (“G-CC” pattern) is integrated (Supplementary Fig. 5c).”

In the paper

“These additional sites did not meet our peak calling criteria, but we detected 2.4% and 1.8% indels at OT1 in two replicates (Supplementary Fig. 8a and Supplementary Table 9), consistent with OT1 being a Cas9:sgFah off-target site in transformed cells in vitro.”

In the paper

“The Cas9 mRNA was present in the liver at 4 h and 14 h but was significantly diminished at 24 h (P < 0.05), consistent with transient expression (Supplementary Fig. 2e).”

In the paper

“We found sgRNA was already expressed at day 3, but its levels were more than tenfold higher at day 7 and day 14.”

In the paper

“The efficiency of correction was >6% of hepatocytes after a single application, suggesting potential utility of Cas9-based therapeutic genome editing for a range of diseases.”

• Also stated in: § Abstract · plain

  “Up to 6.2% ± 1.0% hepatocytes stained positive for the Fah protein by immunohistochemistry in nano.Cas9 plus AAV-HDR–treated animals (Fig. 3b,c; n = 4 mice; see Supplementary Table 4 for percentage of Fah-positive cells, age and gender of each mouse).”

In the paper

“The particle size of nano.Cas9 was the same on day 0, 7, 11 and 18 (Supplementary Fig. 1d,e), indicating these particles are stable for at least 18 d in PBS.”

In the paper

“β-gal protein was detected in the mouse liver by immunoblot at 14 h after administration of a single dose (1 mg/kg or 2 mg/kg), and the amount of protein expressed correlated with the dose of mRNA (Supplementary Fig. 2b).”

In the paper

“Most indels are frameshift (e.g., 1 nt and 2 nt) mutations (Fig. 1e), which potentially cause loss of function of the GFP reporter.”

In the paper

“Systemic delivery of Cas9 mRNA by lipid nanoparticles, and sgRNA/HDR template by AAV, corrected a Fah mutation and restored Fah splicing in more than 6% of hepatocytes in the adult mouse liver, an order of magnitude improvement over that previously generated using high-pressure injection of DNA16.”

What the sources say

• Not checked yetcites [16] Yin, H. et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32, 551–553 (2014). source

In the paper

“This treatment is well-tolerated in mice and fully rescued body weight loss and liver damage in tyrosinemia mice.”

In the paper

“The majority of the cells in liver sections stained positive in a β-gal activity assay (Supplementary Fig. 2c), indicating systemic delivery of mRNA to most of the cells in the mouse liver.”

In the paper

“In HTI patients, mutation of fumarylacetoacetate hydrolase (FAH), the last enzyme catalyzing the tyrosine catabolic pathway, leads to accumulation of toxic metabolites and severe liver damage8.”

What the sources say

• Not checked yetcites [8] Paulk, N.K. et al. Adeno-associated virus gene repair corrects a mouse model of hereditary tyrosinemia in vivo. Hepatology 51, 1200–1208 (2010). source

In the paper

“The Fahmut/mut mouse model6,8 is caused by a G→A point mutation in the last nucleotide of exon 8. This causes skipping of exon 8 and truncated Fah mRNA.”

What the sources say

• Not checked yetcites [6] Aponte, J.L. et al. Point mutations in the murine fumarylacetoacetate hydrolase gene: animal models for the human genetic disorder hereditary tyrosinemia type 1. Proc. Natl. Acad. Sci. USA 98, 641–645 (2001). source
• Not checked yetcites [8] Paulk, N.K. et al. Adeno-associated virus gene repair corrects a mouse model of hereditary tyrosinemia in vivo. Hepatology 51, 1200–1208 (2010). source

In the paper

“An alternative approach is the non-viral delivery of Cas9 mRNA, which would allow for short-term expression and, eventually, complete removal of the nuclease from the body.”

In the paper

“However, all of these delivery studies have reported gene knockout rather than gene repair, a less efficient process requiring a DNA repair template15.”

What the sources say

• Not checked yetcites [15] Chu, V.T., Weber, T. & Wefers, B. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat. Biotechnol. 33, 543–548 (2015). source

In the paper

“Cas9:sgRNA recognizes the protospacer-adjacent motif (PAM) sequence and a complementary 20-nucleotide genomic sequence and induces double-strand DNA breaks, which are repaired by error-prone nonhomologous end-joining (NHEJ) or precise homology-directed repair (HDR)2,5.”

What the sources say

• Not checked yetcites [2] Doudna, J.A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096 (2014). source
• Not checked yetcites [5] Sander, J.D. & Joung, J.K. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355 (2014). source

In the paper

“Recently, a smaller form of Cas9 was developed, and shown capable of packing and delivery with sgRNA by AAV in vivo12.”

What the sources say

• Not checked yetcites [12] Ran, F.A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015). source

In the paper

“Lipid and lipid-like formulations have shown great promise as delivery agents for siRNA in a range of species, including humans19.”

What the sources say

• Not checked yetcites [19] Yin, H. et al. Non-viral vectors for gene-based therapy. Nat. Rev. Genet. 15, 541–555 (2014). source

In the paper

“Nevertheless, hydrodynamic injection yielded a low correction rate of 0.4% of hepatocytes, and this method has been tested in only one human clinical trial17.”

What the sources say

• Not checked yetcites [16] Yin, H. et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32, 551–553 (2014). source
• Not checked yetcites [17] Khorsandi, S.E. et al. Minimally invasive and selective hydrodynamic gene therapy of liver segments in the pig and human. Cancer Gene Ther. 15, 225–230 (2008). source

In the paper

“However, AAV has a loading capacity size of approximately 4.7 kb, and the most commonly used form of Cas9 from Streptococcus pyogenes is difficult to fit in typical AAV constructs with efficient promoters13.”

What the sources say

• Not checked yetcites [13] Swiech, L. et al. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat. Biotechnol. 33, 102–106 (2015). source

In the paper

“These mice can be treated with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), an inhibitor of an enzyme upstream of Fah, to prevent toxin accumulation in hepatocytes8.”

What the sources say

• Not checked yetcites [8] Paulk, N.K. et al. Adeno-associated virus gene repair corrects a mouse model of hereditary tyrosinemia in vivo. Hepatology 51, 1200–1208 (2010). source

In the paper

“To express sgRNA, we treated animals with up to 1.2 × 1012 AAV genome copies/mouse, a standard dose described in the literature28.”

What the sources say

• Not checked yetcites [28] Barzel, A. et al. Promoterless gene targeting without nucleases ameliorates haemophilia B in mice. Nature 517, 360–364 (2015). source

In the paper

“This allowed for short-term expression of the Cas9 nuclease, which provided efficient on-target genome editing, while potentially reducing off-target editing (Fig. 1h).”

In the paper

“A smaller form of Cas9 (Staphylococcus aureus Cas9), which fits into AAV vectors, has been recently used to knockout genes by NHEJ in the mouse liver. More than 40% indel formations in Pcsk9 locus was reported12.”

What the sources say

• Not checked yetcites [12] Ran, F.A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015). source

In the paper

“All animal experiments were performed under the guideline of the MIT Animal Care and Use Committee.”

In the paper

“This estimation is consistent with the 0.81% corrected “G-CC” sequence pattern observed by deep sequencing in treated liver samples (Fig. 3g).”

In the paper

“A recent study demonstrated that the chemical modification of sgRNA improved gene editing in vitro when co-delivered with Cas9 mRNA32.”

What the sources say

• Not checked yetcites [32] Hendel, A. et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol. 33, 985–989 (2015). source

In the paper

“A key feature of our system is the transient expression of the nuclease Cas9, through delivery of mRNA. This allowed for sufficient exposure to the nuclease for gene repair and reduced concerns associated with long-term nuclease exposure34.”

What the sources say

• Not checked yetcites [34] Wu, X. et al. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat. Biotechnol. 32, 670–676 (2014). source

In the paper

“The CRISPR systems have a number of potential advantages over ZFNs, most importantly their flexibility and ease in adjusting target site4.”

What the sources say

• Not checked yetcites [4] Mali, P., Esvelt, K.M. & Church, G.M. Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957–963 (2013). source

In the paper

“However, correction of 6% of hepatocytes following a single round of treatment without selection suggests that this approach may be suitable for the treatment of a range of metabolic liver diseases and hemophilia, where restoration of 3–7% normal functional protein can be therapeutic39.”

What the sources say

• Not checked yetcites [39] Cox, D.B., Platt, R.J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121–131 (2015). source

In the paper

“An alternative explanation for this observation is partial gene conversion in spite of “G” and “CC” single-nucleotide polymorphisms equidistant to the Cas9 cutting site.”

What the sources say

• Not checked yetcites [36] Elliott, B., Richardson, C., Winderbaum, J., Nickoloff, J.A. & Jasin, M. Gene conversion tracts from double-strand break repair in mammalian cells. Mol. Cell. Biol. 18, 93–101 (1998). source
• Not checked yetcites [37] Goldberg, A.D. et al. Distinct factors control histone variant H3.3 localization at specific genomic regions. Cell 140, 678–691 (2010). source
• Not checked yetcites [38] Findlay, G.M., Boyle, E.A., Hause, R.J., Klein, J.C. & Shendure, J. Saturation editing of genomic regions by multiplex homology-directed repair. Nature 513, 120–123 (2014). source

In the paper

“Previous work has reported on the use of zinc finger nucleases (ZFNs) for in vivo gene correction31,33.”

What the sources say

• Not checked yetcites [31] Mahiny, A.J. et al. In vivo genome editing using nuclease-encoding mRNA corrects SP-B deficiency. Nat. Biotechnol. 33, 584–586 (2015). source
• Not checked yetcites [33] Li, H. et al. In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature 475, 217–221 (2011). source

In the paper

“It is possible that our stringent “G-CC” pattern counting underestimated the HDR event.”

In the paper

“Although NHEJ has been reported using AAV-mediated Cas9 delivery, HDR using viral delivery in vivo has not been reported.”

In the paper

“Cells were tested by MIT core facility for mycoplasma contamination by an ELISA-based assay, and confirmed negative.”

In the paper

“For example, less than 10% indels at the Apob locus in the mouse liver was reported in the same study12.”

What the sources say

• Not checked yetcites [12] Ran, F.A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015). source