CRISPR genome engineering for retinal diseases.

Novel gene therapy treatments for inherited retinal diseases have been at the forefront of translational medicine over the past couple of decades. Since the discovery of CRISPR mechanisms and their potential application for the treatment of inherited human conditions, it seemed inevitable that advances would soon be made using retinal models of disease. The development of CRISPR technology for gene therapy and its increasing potential to selectively target disease-causing nucleotide changes has been rapid. In this chapter, we discuss the currently available CRISPR toolkit and how it has been and can be applied in the future for the treatment of inherited retinal diseases. These blinding conditions have until now had limited opportunity for successful therapeutic intervention, but the discovery of CRISPR has created new hope of achieving such, as we discuss within this chapter.

An analysis of the Kozak consensus in retinal genes and its relevance to gene therapy.

Purpose: The classic Kozak consensus is a critical genetic element included in gene therapy transgenes to encourage the translation of the therapeutic coding sequence. Despite optimizations of other transgene elements, the Kozak consensus has not yet been considered for potential tissue-specific sequence refinement. We screened the -9 to -1 region relative to the AUG start codon of retina-specific genes to identify whether a Kozak consensus that is different from the classic sequence may be more appropriate for inclusion in gene therapy transgenes that treat inherited retinal disease. Methods: Sequences for 135 genes known to cause nonsyndromic inherited retinal disease were extracted from the NCBI database, and the -9 to -1 nucleotides were compared. This panel was then refined to 75 genes with specific retinal functions, for which the -9 to -1 nucleotides were placed in front of a GFP transcript sequence and RNAfold predictions performed. These were compared with a GFP sequence with the classic Kozak consensus (GCCGCCACC), and sequences from retinal genes with minimum free energy (MFE) predictions greater than the reference sequence were selected to generate an optimized Kozak consensus sequence. The original Kozak consensus and the refined retina Kozak consensus were placed upstream of the Renilla luciferase coding sequence, which were used to transfect retinoblastoma cell lines Y-79 and WERI-RB-1 and HEK 293T/17 cells. Results: The nucleotide frequencies of the original panel of genes were determined to be comparable to the classic Kozak consensus. RNAfold analysis of a GFP transcript with the classic Kozak sequence in the 5' untranslated region (UTR) generated an MFE prediction of -503.3 kcal/mol. RNAfold analysis was then performed with a GFP transcript containing each -9 to -1 Kozak sequence of 75 retinal genes. Thirty-eight of the 75 genes provided a greater MFE value than -503.3 kcal/mol and exhibited an absence of stable secondary structures before the AUG codon. The -9 to -1 nucleotide frequencies of these genes identified a Kozak consensus of ACCGAGACC, differing from the classic Kozak consensus at positions -9, -5, and -4. Applying this sequence to the GFP transcript increased the MFE prediction to -500.1 kcal/mol. The newly identified retina Kozak sequence was also applied to Renilla luciferase plus the REP1 and RPGR transcripts used in current clinical trials. In all examples, the predicted transcript MFE score increased when compared with the current transcript sequences containing classic Kozak consensus sequences. In vitro transfections identified a 7%-9% increase in Renilla activity when incorporating the optimized Kozak sequence. Conclusions: The Kozak consensus is a critical element of eukaryotic genes; therefore, it is a required feature of gene therapy transgenes. To date, the classic sequence of GCCRCC (-6 to -1) has typically been incorporated in gene therapy transgenes, but the analysis described here suggests that, for vectors targeting the retina, using a Kozak consensus derived from retinal genes can provide increased expression of the target product.

Promoter Orientation within an AAV-CRISPR Vector Affects Cas9 Expression and Gene Editing Efficiency.

Adeno-associated virus (AAV) vectors have been widely adopted for delivery of CRISPR-Cas components, especially for therapeutic gene editing. For a single vector system, both the Cas9 and guide RNA (gRNA) are encoded within a single transgene, usually from separate promoters. Careful design of this bi-cistronic construct is required due to the minimal packaging capacity of AAV. We investigated how placement of the U6 promoter expressing the gRNA on the reverse strand to SaCas9 driven by a cytomegalovirus promoter affected gene editing rates compared to placement on the forward strand. We show that orientation in the reverse direction reduces editing rates from an AAV vector due to reduced transcription of both SaCas9 and guide RNA. This effect was observed only following AAV transduction; it was not seen following plasmid transfection. These results have implications for the design of AAV-CRISPR vectors, and suggest that results from optimizing plasmid transgenes may not translate when delivered via AAV.

CRISPR Interference-Potential Application in Retinal Disease.

The treatment of dominantly inherited retinal diseases requires silencing of the pathogenic allele. RNA interference to suppress gene expression suffers from wide-spread off-target effects, while CRISPR-mediated gene disruption creates permanent changes in the genome. CRISPR interference uses a catalytically inactive 'dead' Cas9 directed by a guide RNA to block transcription of chosen genes without disrupting the DNA. It is highly specific and potentially reversible, increasing its safety profile as a therapy. Pre-clinical studies have demonstrated the versatility of CRISPR interference for gene silencing both in vivo and in ex vivo modification of iPSCs for transplantation. Applying CRISPR interference techniques for the treatment of autosomal dominant inherited retinal diseases is promising but there are few in vivo studies to date. This review details how CRISPR interference might be used to treat retinal diseases and addresses potential challenges for clinical translation.

RNA editing as a therapeutic approach for retinal gene therapy requiring long coding sequences.

RNA editing aims to treat genetic disease through altering gene expression at the transcript level. Pairing site-directed RNA-targeting mechanisms with engineered deaminase enzymes allows for the programmable correction of G>A and T>C mutations in RNA. This offers a promising therapeutic approach for a range of genetic diseases. For inherited retinal degenerations caused by point mutations in large genes not amenable to single-adeno-associated viral (AAV) gene therapy such as USH2A and ABCA4, correcting RNA offers an alternative to gene replacement. Genome editing of RNA rather than DNA may offer an improved safety profile, due to the transient and potentially reversible nature of edits made to RNA. This review considers the current site-directing RNA editing systems, and the potential to translate these to the clinic for the treatment of inherited retinal degeneration.

The Application of CRISPR/Cas9 for the Treatment of Retinal Diseases.

The CRISPR/Cas9 system of genome editing has revolutionized molecular biology, offering a simple, and relatively inexpensive method of creating precise DNA edits. It has potential application in gene therapy treatment of retinal diseases providing targeted disruption, alteration, or transcriptional regulation of pathogenic genes. In vivo studies have demonstrated therapeutic benefit for a variety of diseases. Despite this, there are many challenges to clinical use of CRISPR/Cas9, including editing efficiency, off-target effects, and disease heterogeneity. This review details the mechanisms of the CRISPR/Cas9 system and the treatment strategies that can be applied to retinal diseases. It gives an overview of in vivo studies published to date and discusses the challenges and potential solutions to the wide-scale clinical use of CRISPR/Cas9 as a therapeutic intervention.