Fine-tuning FAM161A gene augmentation therapy to restore retinal function.

For 15 years, gene therapy has been viewed as a beacon of hope for inherited retinal diseases. Many preclinical investigations have centered around vectors with maximal gene expression capabilities, yet despite efficient gene transfer, minimal physiological improvements have been observed in various ciliopathies. Retinitis pigmentosa-type 28 (RP28) is the consequence of bi-allelic null mutations in the FAM161A, an essential protein for the structure of the photoreceptor connecting cilium (CC). In its absence, cilia become disorganized, leading to outer segment collapses and vision impairment. Within the human retina, FAM161A has two isoforms: the long one with exon 4, and the short one without it. To restore CC in Fam161a-deficient mice shortly after the onset of cilium disorganization, we compared AAV vectors with varying promoter activities, doses, and human isoforms. While all vectors improved cell survival, only the combination of both isoforms using the weak FCBR1-F0.4 promoter enabled precise FAM161A expression in the CC and enhanced retinal function. Our investigation into FAM161A gene replacement for RP28 emphasizes the importance of precise therapeutic gene regulation, appropriate vector dosing, and delivery of both isoforms. This precision is pivotal for secure gene therapy involving structural proteins like FAM161A.

Premature Vertebral Mineralization in hmx1-Mutant Zebrafish.

H6 family homeobox 1 (HMX1) regulates multiple aspects of craniofacial development, and mutations in HMX1 are linked to an ocular defect termed oculoauricular syndrome of Schorderet-Munier-Franceschetti (OAS) (MIM #612109). Recently, additional altered orofacial features have been reported, including short mandibular rami, asymmetry of the jaws, and altered premaxilla. We found that in two mutant zebrafish lines termed hmx1mut10 and hmx1mut150, precocious mineralization of the proximal vertebrae occurred. Zebrafish hmx1mut10 and hmx1mut150 report mutations in the SD1 and HD domains, which are essential for dimerization and activity of hmx1. In hmx1mut10, the bone morphogenetic protein (BMP) antagonists chordin and noggin1 were downregulated, while bmp2b and bmp4 were highly expressed and specifically localized to the dorsal region prior to the initiation of the osteogenic process. The osteogenic promoters runx2b and spp1 were also upregulated. Supplementation with DMH1-an inhibitor of the BMP signaling pathway-at the specific stage in which bmp2b and bmp4 are highly expressed resulted in reduced vertebral mineralization, resembling the wildtype mineralization progress of the axial skeleton. These results point to a possible role of hmx1 as part of a complex gene network that inhibits bmp2b and bmp4 in the dorsal region, thus regulating early axial skeleton development.

Hmx1 regulates urfh1 expression in the craniofacial region in zebrafish.

H6 family homeobox 1 (HMX1) regulates multiple aspects of craniofacial development as it is widely expressed in the eye, peripheral ganglia and branchial arches. Mutations in HMX1 are linked to an ocular defect termed Oculo-auricular syndrome of Schorderet-Munier-Franceschetti (MIM #612109). We identified UHRF1 as a target of HMX1 during development. UHRF1 and its partner proteins actively regulate chromatin modifications and cellular proliferation. Luciferase assays and in situ hybridization analyses showed that HMX1 exerts a transcriptional inhibitory effect on UHRF1 and a modification of its expression pattern. Overexpression of hmx1 in hsp70-hmx1 zebrafish increased uhrf1 expression in the cranial region, while mutations in the hmx1 dimerization domains reduced uhrf1 expression. Moreover, the expression level of uhrf1 and its partner dnmt1 was increased in the eye field in response to hmx1 overexpression. These results indicate that hmx1 regulates uhrf1 expression and, potentially through regulating the expression of factors involved in DNA methylation, contribute to the development of the craniofacial region of zebrafish.