Smelling the roses and seeing the light: gene therapy for ciliopathies

Abstract

Alterations in cilia formation or function underlie a growing class of pleiotropic disorders termed ciliopathies. The genetic basis of ciliopathies is remarkably complex, with an incomplete but expanding list of more than 89 loci implicated in various disorders. Current treatment of ciliopathies is limited to symptomatic therapy. However, our growing understanding of ciliopathy genetics, coupled with recent advances in gene delivery and endogenous gene and transcript repair demonstrated thus far in tissues of the eye, nose, and airway, offers hope for curative measures in the near future. This review highlights these advances, as well as the challenges that remain with the development of personalized medicine for treating a very complex spectrum of disease, penetrant in a variety of organ systems.

Figure 1. Gene therapy has been successful in several ciliated cell types

(A) Ciliopathies encompass phenotypes in multiple organ systems that can alter development, impair sensory systems, and lead to tissue degeneration. RTE, renal tubule epithelium. (B) Ciliopathies show pleiotropic penetrance. In the nasal cavity, both olfactory sensory neurons and respiratory epithelium cells possess cilia. Scanning electron micrographs of the OE/RE border show a loss of olfactory cilia, but not respiratory cilia in Bbs4 null mice (Mice provided by Dr. Val Sheffield, University of Iowa). OE, olfactory epithelium; RE, respiratory epithelium; scale bar = 2.5μm. (C) Macrociliary complexes, including IFT, ADPKD, MKS, NPHP and BBS proteins regulate cilia development, maintenance and/or function. Mutations in genes encoding proteins involved in distinct complexes often share similarities in phenotypes. Inset depicts vesicular accumulation along the ciliary axoneme in Bbs4 mutant mice [79]. (D) Primary and motile cilia posses distinct axonemal microtubule structures. Disorganization of these structures can cause PCD, through defects in ciliary motility. MT, microtubule. (E) To date, cilium-focused gene therapy strategies have been successful in several cell types, including retinal photoreceptors (in both human and mouse models), respiratory epithelial cells (ex vivo), and olfactory sensory neurons (in mouse models).

Figure 2. Strategies for rescuing mutant genes

(A) Ciliopathy mutations can lead to altered gene expression or protein function through insertions/deletions or point mutations that disrupt mRNA splicing, cause premature termination, or change protein coding. (Bottom, right) Representation of a ciliopathy gene mutation causing protein truncation and disruption of a ciliary functional unit. (B) (Right) To date, delivery of genes in ciliopathy models has been mediated by adeno-associated virus (AAV), lentivirus, and adenovirus (AV). (Left) Delivery methods of gene therapies to the visual and olfactory systems (via retinal injection and intranasal delivery, respectively) are minimally invasive. Urethral injection of the kidney represents a feasible delivery method for treating ciliopathies affecting internal organs. (C) Viral vectors can be used to express (top) whole genes in targeted cells. (Bottom) In the event of a full gene surpassing the size limitations imposed by viral packaging, a partial gene fragment could potentially be used to specifically replace the missing portion of a truncated protein. (D) Mutations that occur in splice donor sites can prevent complex formation of the splice factor U1 snRNP with pre-mRNAs and result in (left) intronic read-through or exon skipping. Viral expression of a modified U1 snRNA, to increase its complementarity to the mutated splice site, can correct misspliced transcripts allowing expression of the full-length gene. Sequences shown correspond to the splice donor site of BBS1 exon/intron 5 affected by the c.479G-A mutation and the modified U1 snRNP virally expressed to restore splicing as reported in [41]. (E) Finally, targeted ciliopathy gene repair/genome editing machineries have been successfully delivered by viral mechanisms. Zinc-finger nucleases (ZFNs), encoded by virally delivered minigenes and fused to FokI nuclease, target genomic DNA surrounding a ciliopathy mutation and induce a double-stranded DNA break. Subsequent homologous recombination with a correct version of the sequence (presented via gene delivery) results in a repaired genomic locus.