Developing new ways of delivering cells to diseased tissue will be a key factor in translating cell therapeutics research into clinical use. Magnetically targeting cells enables delivery of significant numbers of cells to key areas of specific organs. To demonstrate feasibility in neurological tissue, we targeted cells magnetically to the upper hemisphere of the rodent retina. Rat mesenchymal stem cells (MSCs) were magnetized using superparamagnetic iron oxide nanoparticles (SPIONs). In vitro studies suggested that magnetization with fluidMAG-D was well tolerated, that cells remained viable, and they retained their differentiation capabilities. FluidMAG-D-labeled MSCs were injected intravitreally or via the tail vein of the S334ter-4 transgenic rat model of retinal degeneration with or without placing a gold-plated neodymium disc magnet within the orbit, but outside the eye. Retinal flatmount and cryosection imaging demonstrated that after intravitreal injection cells localized to the inner retina in a tightly confined area corresponding to the position of the orbital magnet. After intravenous injection, similar retinal localization was achieved and remarkably was associated with a tenfold increase in magnetic MSC delivery to the retina. Cryosections demonstrated that cells had migrated into both the inner and outer retina. Magnetic MSC treatment with orbital magnet also resulted in significantly higher retinal concentrations of anti-inflammatory molecules interleukin-10 and hepatocyte growth factor. This suggested that intravenous MSC therapy also resulted in significant therapeutic benefit in the dystrophic retina. With minimal risk of collateral damage, these results suggest that magnetic cell delivery is the best approach for controlled delivery of cells to the outer retina-the focus for disease in age-related macular degeneration and retinitis pigmentosa.

Nonsense mutations leading to premature stop codons are common occurring in approximately 12% of all human genetic diseases. Thus, pharmacological nonsense mutation suppression strategies would be beneficial to a large number of patients if the drugs could be targeted to the affected tissues at the appropriate time. Here, we used nonsense suppression to manipulate Pax6 dosage at different developmental times in the eye of the small eye (Pax6Sey/+; G194X) mouse model of aniridia. Efficacy was assessed by functional assays for visual capacity, including electroretinography and optokinetic tracking (OKT), in addition to histological and biochemical studies. Malformation defects in the Pax6Sey/+ postnatal eye responded to topically delivered nonsense suppression in a dose- and time-dependent manner. Elevated levels of Mmp9, a direct downstream target of Pax6 in the cornea, were observed with the different treatment regimens. The lens capsule was particularly sensitive to Pax6 dosage, revealing a potential new role for Pax6 in lens capsule maintenance and development. The remarkable capacity of malformed ocular tissue to respond postnatally to Pax6 dosage in vivo demonstrates that the use of nonsense suppression could be a valuable therapeutic approach for blinding diseases caused by nonsense mutations.

Mutations in KIF14 have previously been associated with either severe, isolated or syndromic microcephaly with renal hypodysplasia (RHD). Syndromic microcephaly-RHD was strongly reminiscent of clinical ciliopathies, relating to defects of the primary cilium, a signalling organelle present on the surface of many quiescent cells. KIF14 encodes a mitotic kinesin, which plays a key role at the midbody during cytokinesis and has not previously been shown to be involved in cilia-related functions. Here, we analysed four families with fetuses presenting with the syndromic form and harbouring biallelic variants in KIF14. Our functional analyses showed that the identified variants severely impact the activity of KIF14 and likely correspond to loss-of-function mutations. Analysis in human fetal tissues further revealed the accumulation of KIF14-positive midbody remnants in the lumen of ureteric bud tips indicating a shared function of KIF14 during brain and kidney development. Subsequently, analysis of a kif14 mutant zebrafish line showed a conserved role for this mitotic kinesin. Interestingly, ciliopathy-associated phenotypes were also present in mutant embryos, supporting a potential direct or indirect role for KIF14 at cilia. However, our in vitro and in vivo analyses did not provide evidence of a direct role for KIF14 in ciliogenesis and suggested that loss of kif14 causes ciliopathy-like phenotypes through an accumulation of mitotic cells in ciliated tissues. Altogether, our results demonstrate that KIF14 mutations result in a severe syndrome associating microcephaly and RHD through its conserved function in cytokinesis during kidney and brain development.

Achromatopsia is a progressive autosomal recessive retinal disease characterized by early loss of cone photoreceptors and later rod photoreceptor loss. In most cases, mutations have been identified in CNGA3, CNGB3, GNAT2, PDE6C or PDE6H genes. Owing to this genetic heterogeneity, mutation-independent therapeutic schemes aimed at preventing cone cell death are very attractive treatment strategies. In pde6c w59 mutant zebrafish, cone photoreceptors expressed high levels of receptor-interacting protein kinase 1 (RIP1) and receptor-interacting protein kinase 3 (RIP3) kinases, key regulators of necroptotic cell death. In contrast, rod photoreceptor cells were alternatively immunopositive for caspase-3 indicating activation of caspase-dependent apoptosis in these cells. Morpholino gene knockdown of rip3 in pde6c w59 embryos rescued the dying cone photoreceptors by inhibiting the formation of reactive oxygen species and by inhibiting second-order neuron remodelling in the inner retina. In rip3 morphant larvae, visual function was restored in the cones by upregulation of the rod phosphodiesterase genes (pde6a and pde6b), compensating for the lack of cone pde6c suggesting that cones are able to adapt to their local environment. Furthermore, we demonstrated through pharmacological inhibition of RIP1 and RIP3 activity that cone cell death was also delayed.Collectively, these results demonstrate that the underlying mechanism of cone cell death in the pde6c w59 mutant retina is through necroptosis, whereas rod photoreceptor bystander death occurs through a caspase-dependent mechanism. This suggests that targeting the RIP kinase signalling pathway could be an effective therapeutic intervention in retinal degeneration patients. As bystander cell death is an important feature of many retinal diseases, combinatorial approaches targeting different cell death pathways may evolve as an important general principle in treatment.

Tissue fusion is an essential morphogenetic mechanism in development, playing a fundamental role in developing neural tube, palate and the optic fissure. Disruption of genes associated with the tissue fusion can lead to congenital malformations, such as spina bifida, cleft lip/palate and ocular coloboma. For instance, the Pax2 transcription factor is required for optic fissure closure, although the mechanism of Pax2 action leading to tissue fusion remains elusive. This lack of information defining how transcription factors drive tissue morphogenesis at the cellular level is hampering new treatments options. Through loss- and gain-of-function analysis, we now establish that pax2 in combination with vax2 directly regulate the fas-associated death domain (fadd) gene. In the presence of fadd, cell proliferation is restricted in the developing eye through a caspase-dependent pathway. However, the loss of fadd results in a proliferation defect and concomitant activation of the necroptosis pathway through RIP1/RIP3 activity, leading to an abnormal open fissure. Inhibition of RIP1 with the small molecule drug necrostatin-1 rescues the pax2 eye fusion defect, thereby overcoming the underlying genetic defect. Thus, fadd has an essential physiological function in protecting the developing optic fissure neuroepithelium from RIP3-dependent necroptosis. This study demonstrates the molecular hierarchies that regulate a cellular switch between proliferation and the apoptotic and necroptotic cell death pathways, which in combination drive tissue morphogenesis. Furthermore, our data suggest that future therapeutic strategies may be based on small molecule drugs that can bypass the gene defects causing common congenital tissue fusion defects.