Gene editing with CRISPR/Cas9 is here, there, and everywhere. We have seen gene editing in human somatic cells, pluripotent stem cells, and even embryos. However, we should also be looking at CRISPR-based gene editing somewhere else: in a living, breathing human. The question is – where does the current technology stand on the in vivo treatment of genetic diseases through the application of CRISPR/Cas9?
First in vivo CRISPR/Cas9 Correction
The first application came in 2014 from the laboratory of Daniel G Anderson. They aimed to correct a mutation in the fumarylacetoacetate hydrolase (Fah) gene in liver cells of a mouse model of hereditary tyrosinemia type I (HTI) . Their “simple” strategy involved the hydrodynamic tail vein injection of a plasmid vector co-expressing an Fah-specific single guide (sg)RNA, Cas9, and a single stranded DNA donor to facilitate homologous recombination and correction of the mutation.
This prevented the weight loss and liver damage normally observed in this model, and the authors linked this functional rescue to the re-expression of wild-type Fah in hepatocytes (implying mutation correction) and the in vivo expansion of this initially small cell population. Although the study found no notable off-target events (from predicted off-target sites), the rate of correction required further enhancements, and the delivery strategy was not fully compatible with clinical transference. So while this stood as proof of principle, we needed something better, or in fact, something smaller!
Cas9 – Smaller is Better
One of the best ways to target as many cells as possible for systemic gene-editing delivery is the versatile adeno-associated virus (AAV). However, the Streptococcus pyogenes Cas9 (SpCas9) that scientists had been using was too large to squeeze inside this small vector. Feng Zhang and colleagues set off on the hunt for a smaller, effective Cas9, and soon found that Cas9 from Staphylococcus aureus (SaCas9), at about 1 kb smaller, was ideal for AAV-mediated in vivo gene editing .
They packaged their new discovery alongside an sgRNA expression cassette designed to target and disrupt the Pcsk9 cholesterol regulatory gene in mouse liver following tail vein injection. While not used to correct a mutation, the disruption of this Pcsk9 did mediate a significant decrease in total levels of cholesterol – a great finding for those suffering from an over-abundance of cholesterol and the associated problems. Furthermore, genome-wide analysis using BLESS (direct in situ breaks labelling, enrichment on streptavidin and next-generation sequencing) in liver tissue found very low off-target activity, and they observed no signs of an abnormal response to the AAV in the liver, suggesting that this strategy was both specific and safe.
Triple Triumph for Duchenne Muscular Dystrophy (DMD) Treatment in Adults
Amy J. Wagers, Charles A. Gersbach, and Eric N. Olson all saw the great promise of systemic gene editing using the AAV-system, and they all hit on the same idea – the creation of a viable treatment for patients suffering Duchenne Muscular Dystrophy (DMD). This rare recessive X-linked form of muscular dystrophy results in muscle degeneration and premature death and is caused by mutations in the very large human Dystrophin gene (all 79 exons of it!) that cause genetic frame-shifts or loss of protein expression.
One potential treatment strategy is to remove some of the affected regions to generate a truncated, but still functional, Dystrophin protein. Many studies have shown this to be a viable treatment strategy (very little Dystrophin is actually required), but they had only described this in isolated muscle cells, induced pluripotent stem cells, or in the mouse germline. Now, three papers in Science [3-5] describe the successful application of this strategy in the adult mdx mouse model of human DMD using CRISPR/Cas9.
Two of the studies [3, 4] found that local and systemic delivery of AAVs laden with an SaCas9 designed to excise the affected exons restored Dystrophin expression in myofibers, cardiomyocytes, and muscle stem cells. This led to the partial recovery of muscle functional deficiencies, including improvement of muscle biochemistry and significant enhancement of muscle force. Furthermore, the study detected no off-target effects, and the mice tolerated the treatment well. Indeed, the studies showed that one-time administrations were sufficient to generate enough Dystrophin protein to suppress the dystrophic phenotype.
The third study  also used an AAV, although they employed a humanized version of SpCas9 to do the editing. The results, however, were the same: injection into the musculature mediated the re-expression of Dystrophin in adult mice and enhanced skeletal muscle function.
So, the animal models show that the strategy works, how long till human trials?
Hitting Gain-of-Function Mutations
Most of what we think of when we talk of gene replacement is compensating for loss-of-function mutations. However, some diseases deal with an unfortunate gain-of-function mutation whose actions generate all the damage. One of these diseases is severe autosomal dominant Retinitis Pigmentosa (adRP) caused by a monoallelic, gain-of-function mutation in the Rhodopsin gene.
The key term there is monoallelic, meaning that if we can get rid of the pesky mutant allele, the wild-type allele can step up and functionally compensate. Enter CRISPR/Cas9 and a study from Shaomei Wang in a rat model of adRP . They showed that a single subretinal injection of gRNA/SpCas9 plasmid followed by electroporation (sounds sore!) mediated the allele-specific disruption of the mutation, prevented retinal degeneration, and improved visual function.
A similar strategy was also used in another study from Andrew Nesbit and Tara Moore in the treatment of Meesmann’s epithelial corneal dystrophy (MECD)  caused by a dominant-negative mutation in the keratin 12 (KRT12) gene. Intrastromal ocular injection of a combined sgRNA/SpCas9 expression construct into a humanized MECD mouse model caused non-homologous end-joining repair at the mutant gene, resulting in frame-shifting deletions. The authors note that while this was partially effective, they required an increase in the targeting efficiency (again noting in vivo electroporation as a strategy) to achieve enough of a knockdown to reverse or inhibit disease pathology.
Not just Genetic Correction, but Epigenetic Correction
Finally, we move on to a different means of CRISPR-mediated treatment – reversing epigenetic dysregulation. This time, Peter and Takako Jones attempted to use catalytically inactive forms of Cas9 (dCas9) to reverse the epigenetic derepression of the D4Z4 macrosatellite repeat array observed in those suffering from Facioscapulohumeral muscular dystrophy (FSHD) . Dysregulation leads to the aberrant expression of D4Z4-encoded RNAs in skeletal muscle which results in muscle dysfunction.
Their new study used sgRNAs and a dCas9 fused to the KRAB transcriptional repressor to target the affected site and alter the chromatin environment (rather than actually “nick” the DNA or correct any mutation) to re-repress the affected region in primary FSHD myocytes. Encouragingly, after infection with dCas9-KRAB, they found elevated levels of heterochromatin-associated proteins (KAP1/TRIM28 co-repressor and HP1a and b) and downregulation of the mis-expressed RNAs. Hopefully, this seemingly effective in vitro treatment will be extended to an in vivo assessment in the near future.
The Future of CRISPR/Cas9
The recent flurry of exciting studies is surely only a prelude to the publication of more and more effective and precise CRISPR/Cas9-based treatment strategies for a swath of genetic diseases in animal models. This will also be accompanied by further enhancement of the tools we have at our disposal, as exemplified by the Cas9 “arms race” currently playing itself out in the literature (See the Nature and Science papers for the details)! Will these two research lines combine to lead to a clinical trial in human patients in the near future? Keep tuned to Epigenie to find out.
- Yin H, Xue W, Chen S, et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat Biotechnol 2014;32:551-553.
- Ran FA, Cong L, Yan WX, et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 2015;520:186-191.
- Tabebordbar M, Zhu K, Cheng JK, et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science 2015; In Press
- Nelson CE, Hakim CH, Ousterout DG, et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science 2015; In Press.
- Long C, Amoasii L, Mireault AA, et al. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science 2015; In Press.
- Bakondi B, Lv W, Lu B, et al. In Vivo CRISPR/Cas9 Gene Editing Corrects Retinal Dystrophy in the S334ter-3 Rat Model of Autosomal Dominant Retinitis Pigmentosa. Mol Ther 2015; In Press.
- Courtney DG, Moore JE, Atkinson SD, et al. CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting. Gene Ther 2016;23:108-112.
- Himeda CL, Jones TI, and Jones PL CRISPR/dCas9-mediated Transcriptional Inhibition Ameliorates the Epigenetic Dysregulation at D4Z4 and Represses DUX4-fl in FSH Muscular Dystrophy. Mol Ther 2015; In Press.