Retinal degeneration is an irreversible process that ultimately leads to blindness and preservation of existing vision is a promising therapeutic strategy. Ciliary neurotrophic factor (CNTF) and OncostatinM (OSM) are two potent survival factors for neurons and oligodendrocytes. However, growth factors degrade quickly, and re-injections are needed to maintain its protective role. A stable delivery of these growth factors is required for translating its therapeutics into patients.
Stable and biocompatible nanoparticles incorporated with CNTF and OSM (NP-CNTF and NP-OSM) were produced. To evaluate their neuroprotective effects, a novel in vitro assay platform using retinal photoreceptor precursor (RPP) cells and retinal ganglion progenitor cells (RGPC) derived from human induced pluripotent stem cells (iPSC) was developed. To investigate their neuroprotective effects in vivo, a single dose of NP-CNTF, NP-OSM, or control NPs were injected intravitreally in the Royal College of Surgeons (RCS) rats (n=6/group), a well-established rodent model for RP and electroretinography (ERG) and optokinetic response (OKR) were carried out at postnatal day 60 and 90 (P60 and P90). In addition, intravitreal injection of NP-CNTF or NP-OSM into an optic nerve crush (ONC) model (n=6) to examine retinal ganglion cell (RGC) survival. Retinal histology was performed at the end of experiments.
Significant pro-survival and pro-proliferation effects of both complexes were observed in both iPSC-RPP and iPSC-RGPC platforms within a broad range of doses (from 0.2 to 20 ng/ml). Significant preservation of vision as tested by ERG and OKR in NP-OSM treated animals as compared with other groups. However, both NP-CNTF/OSM showed significant RGC protection compared with controls in ONC model. Histological analyses showed a global photoreceptor preservation in NP-OSM treated RCS rats, dramatically different from NP-CNTF and controls. More importantly, an intact RPE layer was observed in NP-OSM treated retina, which is in contrast tohypertrophic and disorganized RPE layer observed in control P90 RCS retina.
Our in vitro and in vivo results demonstrated that these stable and biocompatible NPFs were able to:
1. Promote the survival and proliferation of retinal neurons in vitro;
2. Protect retinal ganglion cells in a rat optical nerve crush model;
3. Preserve visual functions in RCS rats long term;
4. Prevent retinal photoreceptor degeneration;
5. Preserve the integrity of the important RPE layer;
6. Halt/slow down degeneration of the whole retina in RCS rats.
These results demonstrate that stable and biocompatible NP-OSM /CNTF are neuroprotective both in vitro and in vivo, indicating a strong therapeutic value for retinal neurodegeneration diseases and other neurodegenerative diseases as well. The same approach can be extended to other factors for the prevention/treatment of other degenerative diseases.
Illustration of nanoparticles incorporated with protein factors
Both NPFs and native proteins promoted the survival and proliferation of retinal photoreceptor progenitor cells derived from three (3) different human iPSC lines with similar efficacy.
Upper: Both NPFs and native proteins promoted the survival and proliferation of human iPSC-derived retinal ganglion progenitor cells with similar efficacy;
Lower: Both NPFs protected retinal ganglion cells from degeneration after optical nerves were crushed in a rat model.
Visual function tested by OKR showed significantly preservation in NP-NPFs treated eyes compared with control groups (BSS and untreated) in RCS rats at both P60 and P90.
Visual function tested by ERG showed significantly preservation in NP-NPFs treated eyes compared with control groups (BSS and untreated) in RCS rats at both P60 and P90.
NP-NPFs treatment globally prevented photoreceptors from degeneration (8-10 layers (C & D) vs 1-2 layers (A), upper), and it also preserved the integrity of retina—both photoreceptors (recoverin, red) and RPE (RPE65, green) were preserved at P90 after NP-NPFs treatment in RCS rats.