CRISPR-Cas9 genome engineering: Treating inherited retinal degeneration

Abstract

Gene correction is a valuable strategy for treating inherited retinal degenerative diseases, a major cause of irreversible blindness worldwide. Single gene defects cause the majority of these retinal dystrophies. Gene augmentation holds great promise if delivered early in the course of the disease, however, many patients carry mutations in genes too large to be packaged into adeno-associated viral vectors and some, when overexpressed via heterologous promoters, induce retinal toxicity. In addition to the aforementioned challenges, some patients have sustained significant photoreceptor cell loss at the time of diagnosis, rendering gene replacement therapy insufficient to treat the disease. These patients will require cell replacement to restore useful vision. Fortunately, the advent of induced pluripotent stem cell and CRISPR-Cas9 gene editing technologies affords researchers and clinicians a powerful means by which to develop strategies to treat patients with inherited retinal dystrophies. In this review we will discuss the current developments in CRISPR-Cas9 gene editing in vivo in animal models and in vitro in patient-derived cells to study and treat inherited retinal degenerative diseases.

Keywords: CRISPR-Cas9; Genome engineering; Induced pluripotent stem cells; Retinal degeneration; Transplantation.

Figure 1.. The CRISPR-Cas systems function in bacteria and archea as adaptive immune systems against foreign genetic material.

The system is composed of a CRISPR array of alternating conserved repeats and target-specific spacers (protospacers) acquired from fragments of foreign genetic material. The bacterium creates a heritable memory of infection. Upon entry of a foreign invader (1 – infection), foreign DNA sequences are incorporated into the bacterial CRISPR locus (2 – acquisition) and subsequently the bacterium transcribes CRISPR RNAs (crRNAs) from the array which associate with Cas effector proteins to create a ribonucleoprotein surveillance complex (RSC) (3 – expression). The RSC recognizes a sequence directly downstream of the crRNA target sequence – the protospacer adjacent motif (PAM). Following guide binding, the Cas nuclease cleaves the target DNA leading to the clearance of the foreign invader (4 – interference) (Leenay and Beisel, 2017).

Figure 2.. AAV serotype transduction comparison following subretinal injection in mouse retina.

A) Schematic digram depicting the layers of the neural retinal and placement of AAV vectors in vivo. B-H) Immunohistochemical analysis of GFP expression (green) driven by seven different AAV serotypes two weeks post-subretinal injection. Representative z-stacks are shown for each of AAV1 (B-B’), AAV2 (C-C’), AAV4 (D-D’), AAV5 (E-E’), AAV6 (F-F’), AAV8 (G-G’) and AAV9 (H-H’). DAPI was used to visualize retinal nuclei. NFL – nerve fiber layer, GCL – ganglion cell layer, IPL – inner plexiform layer, INL – inner nuclear layer, OPL – outer plexiform layer, ONL – outer nuclear layer, IS/OS – photoreceptor cell inner and outer segments. Scale bars = 50 μm.

Figure 3.. Efficency of CRISPR based genome editing.

A-B: Immunohistochemical analysis of GFP expression (green) following transduction of human retinal explants with AAV5-GFP at 1 week post-subretinal delivery. Unlike in vitro HEK293 transfection efficiency, which is near 100%, AAV5 based gene delivery vectors typically transduce less that 50% of the photoreceptor cells targeted. C: In vitro NHEJ efficiency. Of 40 sgRNAs targeting ten independent genes associated with inherited retinal degenerative disease, an average NHEJ efficiency of 20.6 ± 1.3% NHEJ was detected.

Figure 4.. Human retinal tissue engineering.

The inner retina, which is preserved during inherited retinal degeneration, and the photoreceptor cell layer are intimately connected (schematic shown in A). The outer nuclear layer (ONL) comprises photoreceptor cell bodies stacked in columns while the outer segments are tightly packed side-by-side in close contact with the RPE (not shown). When viewed en face via phase contrast microscopy (B), human photoreceptor cells appear very tightly packed in a hexagonal array. This architecture can be closely recapitulated using high-resolution 3D printing (C-E), enabling the creation of photoreceptor cell scaffolds (F-H) meant to enable proper function and integration with the host inner retina. These highly tunable structures can facilitate close cell packing, guide cell orientation, and release small molecules that encourage synaptogenesis (I). GCL – ganglion cell layer, IPL – inner plexiform layer, INL – inner nuclear layer, OPL – outer plexiform layer, ONL – outer nuclear layer, IS/OS – photoreceptor cell inner and outer segments.