Virus-based methods are widely used in the mammalian nervous system for expressing genes (1) and for producing short hairpin RNAs (shRNAs) (2) to knock down genes by RNA interference (RNAi). In the latter case, only a percentage of the small interfering RNAs (siRNAs) can effectively silence their cognate target genes (3). Thus, multiple selections of siRNA are often required. This multiplicity can make the virus-based method time-, labor-, or cost-intensive, especially when compounded with the goal of targeting multiple genes. Also, not all neuronal types are susceptible targets of a viral carrier. For example, retinal ganglion cells (RGCs) are known so far to be readily transduced only by adeno- associated virus and lentiviruses (4,5,6). Here, we report the successful use of a polymer as a carrier to deliver shRNA- expressing plasmid DNA to these cells in vivo.

Nonviral carriers such as polymers are simple to use and can be safer than viral carriers. The cationic polymer polyethylenimine (PEI) has been used for transfection in vitro and in vivo mostly of non-neuronal tissues (7). We tested the efficiency of PEI/DNA polyplexes for transfecting RGCs in vivo, so chosen because these cells are adjacent to the vitreous and therefore are likely accessible to the polyplexes delivered by intravitreal injection.

A commercial vector (RNAi-Ready pSIREN-DNR-DsRed-Express; Clontech, Mountain View, CA, USA) expressing shRNA (driven by a human U6 promoter) and a reporter Discosoma red fluorescent protein (DsRed) [driven by a cytomegalovirus (CMV) promoter] was mixed with PEI (in vivo-jetPEI™; Polyplus Transfection, Illkirch, France) according to the manufacturer's instructions. In this study, the N:P ratio (i.e., the number of nitrogen residues of in vivo-jetPEI per DNA phosphate) used was 10 (e.g., 1 µg DNA was mixed with 0.2 µL in vivo-jetPEI). The PEI/DNA polyplex solution (1.2 µL for each eye) was carefully administered intravitreally (Figure 1A) from the posterior-temporal side of the eye of an anesthetized mouse via a no. 33 custom needle (1-inch-long/sharp point/type no. 2; Hamilton, Reno, NV, USA) on a 2.5-µL Hamilton syringe. The resulting expression of DsRed was evident in many cells in the ganglion cell layer (Figure 1B). In the retinal cross-section, DsRed expression was confirmed to be in the ganglion cell layer, indicating that the intravitreally injected polyplexes were able to cross the optic nerve fiber layer (containing the axons of the ganglion cells) from the vitreous (Figure 1C).

Figure 1. In vivo polymer-mediated gene delivery in retina. (Click to enlarge)

We next examined the shRNA expression. We designed the shRNA to target melanopsin (8), the photopigment mediating the light response of the intrinsically photosensitive RGCs (ipRGCs) (9,10). The intrinsic photosensitivity of the ipRGCs is required in order for the pupillary light reflex to reach completion at high irradiances, with melanopsin-knockout mice showing an incomplete pupil restriction in bright light (9). We hypothesized that knocking down melanopsin in the wild-type mouse retina should produce a similar phenotype. We chose an albino background (Balb/c) because the pupil size of melanopsin-knockout mouse with this background (B6.129.Balb/c) at high irradiances was significantly larger than that with a pigmented background (B6.129; unpublished observation). The underlying mechanism for this difference is unclear, but may reflect multiple defects associated with the albino locus, including abnormal axonal projections from the eye to the brain, an underdeveloped central retina, and a deficit of the rod system (11). The pupil reflex in an albino background thus gave a more dramatic indication of melanopsin knockdown. In Figure 2A, immunohistochemistry with a melanopsin antibody (12) indicated that melanopsin expression in the transfected (DsRed positive) area was reduced to an undetectable level by the melanopsin-specific shRNA. In the untransfected (DsRed negative) area of the same retina, melanopsin expression was normal. The eyes injected with melanopsin-shRNA-expressing plasmid DNA showed an incomplete pupil constriction in bright light (10,000 lux), whereas the eyes injected with control DNA were unaffected (Figure 2, B and C). The variation in pupil constriction from eye to eye due to melanopsin-specific shRNA (Figure 2C) conceivably resulted from the variation in the size of the transfected area. Over the entire retina, the average number of remaining melanopsin-immunoreactive cells (all found outside the transfected area) at 5 days after transfection was 176 ± 28 (mean ± SEM; 12 retinas from 6 animals), which translated to 25% ± 4% of the total melanopsin-espressing RGCs (MOP-RGCs) (12). Thus, the average transfected area was 75% ± 4% (mean ± SEM). The variation in the transfected area presumably reflects the technically challenging intravitreal injection into the very limited space between the retina and the lens in mouse. In other mammals, including primates, the intra-vitreal injection should be considerably easier. Part of the variation in pupil size, which has autonomic input from the nervous system (9), could also have come from the animal's stress level during handling (13). Nonetheless, the melanopsin-knockdown effect was clear. The DsRed expression and the melanopsin-knockdown effect started to appear as early as 16 h after injection and lasted at least 2 months (Figure 2D).

(Click to enlarge)

We thus have demonstrated a fast and simple nonviral method using a polymer for delivering DNA to RGCs. From sequence design to injection, it can be as fast as a few days in the case of an RNAi experiment involving even multiple constructs. Recently, it has been shown possible to restore retinal photosensitivity in mice that have degenerated rods and cones by the virus-mediated expression of channelrhodopsin, a photosensitive ion channel, in RGCs (14). Thus, there is considerable research interest in vision reviving gene therapy involving these cells. The current method provides a simple alternative approach. Conceivably, with injection into the subretinal space, the same method can be used for gene delivery to the rods and cones, another active area of research on ameliorating loss of vision associated with defects in rod/cone function (15). As mentioned earlier, the polymer method becomes particularly expedient when the deliveries of many DNA constructs have to be tested or made.