After characterizing eight such mini-PCDH15 versions in vitro and optimizing AAV expression cassettes for inner ear hair cells, we tested their function in vivo in three USH1F mouse models. In this work, we used our structural knowledge of PCDH15 to eliminate 3–5 EC repeats while retaining PCDH15 function, in order to package the coding sequence into a single AAV capsid. PCDH15-CD2 has a ~5.3 kb coding sequence, too large for an AAV capsid. PCDH15 has three alternatively spliced C-termini: CD1, CD2 and CD3, but only the CD2 splice form is necessary for PCDH15 function in hair cells in mice 23, 25. USH1F represents roughly 3–11% of all Usher syndrome cases 36, 37. Additionally, USH1 patients develop prepubertal retinitis pigmentosa (RP), which progresses to nearly complete blindness by the fifth decade 34, 35. Patients with the most severe forms of Usher syndrome, known as type 1 (USH1), experience profound congenital hearing loss and severe balance deficits. It is both genetically and clinically heterogeneous. Usher syndrome (USH) is the most common cause of deaf-blindness worldwide. In humans, mutations in PCDH15 can cause isolated deafness or Usher syndrome type 1 F (USH1F). It also forms transient lateral and kinocilial links, critical for hair bundle development and planar cell polarity 17, 21, 25, and is expressed in retinal photoreceptors and in auditory cortex interneurons 32, 33. Serving as the lower portion of the tip link in mature inner ear hair cells, PCDH15 binds to cadherin 23 (CDH23) 22, 26 to convey force from sound stimuli to the mechanosensory transduction channels 30, 31. This protein is a large, atypical cadherin with 11 link-like extracellular cadherin (EC) repeats and a membrane adjacent domain (MAD12) preceding its single transmembrane domain 22, 29. Over the past decade, we and others have elucidated the molecular structure, function, and interactome of PCDH15 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. However a significant challenge for minigenes is the need to understand the protein structure and binding partners well enough to delete non-essential domains while preserving function, especially when function may include resisting large forces. More recently, mini-otoferlin genes have been used to probe the role of the protein in hair-cell endocytosis in the cochlea 13, 14, mini- CEP290 genes have been evaluated for treating CEP290-associated Leber congenital amaurosis 15, and a “condensed” form of human tuberin (cTuberin) designed for treating tuberous sclerosis complex 16. This strategy has been employed to develop mini- and micro-dystrophins to treat Duchenne muscular dystrophy, based on the observation that large deletions in the dystrophin gene are generally well tolerated 12. Dual and triple AAV approaches are options for circumventing this drawback however they rely on co-transduction of multiple viral particles in the same cell, and on efficient intracellular recombination of the viral DNA or expressed proteins 9, 10, 11.Ī less common approach is to use rational protein design to generate shortened versions of the protein of interest. Elements such as promoters, polyadenylation sequences, and other regulatory elements further limit the size of transgenes that can be packaged. However AAVs remain limited by their capsid packaging capacity, which only allows transport of ~5 kb of genetic material. Capsid design and discovery efforts have dramatically improved AAV transduction rates, particularly in neuronal tissues 1, 2, 3, 4, 5, 6, 7, 8. Furthermore, various AAV serotypes and variants display broad but distinct tropisms that allow targeting to specific cell types 4, 5, 6, 7. Their low toxicity and immunogenicity minimize adverse reactions and they produce long-term transgene expression. Advances in gene therapy have positioned adeno-associated viruses (AAV) as a ubiquitous delivery tool for treating monogenic inherited disorders 1, 2, 3.
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