Regenerative nanomedicine and the treatment of degenerative retinal diseases

Adeno‐associated virus capsid shuffling and directed evolution. Although the capsid sequences can be easily modified, it is difficult to make predictions about how specific modifications in the amino acid sequence will affect the transduction parameters of the viral vector. (a) Various capsid DNA sequences are derived from adeno‐associated viruses (AAVs) with different transduction properties (hexagons of different colors). (b) The capsid DNA sequences are randomly digested and then PCR ligated back into a ‘wild‐type’ AAV plasmid (capS, shuffled cap gene). The AAV capsid library can contain between 10⁶ and 10⁷ unique sequences. (c) The recombinant AAV wild‐type viruses are expanded (with the addition of a replication helper virus, not shown) without any selection in cells. (d) The AAV viral library is expanded under selective pressure, allowing viruses that survive the selection to be further propagated. With stronger selective pressure, the diversity of the capsid library is reduced, and select clones are enriched. (e) Selected capsid sequences that survive the selection are then cloned into a vector production system and used to pseudotype standard AAV vector genomes (containing a reporter or therapeutic expression cassette) and tested for transduction properties in cells, animals, or humans. (Reprinted with permission from Ref 96. Copyright 2011 Nature Publishing Group)

Immunohistochemistry of the retina of rats with EAU (n = 4, each group). Representative results are shown 7 days after treatment with saline (a), nonstealth nanosteroids [poly(lactic acid) (PLA)] (b), and stealth nanosteroids [poly(lactic acid)–poly(ethylene glycol) (PLA–PEG)] (c). Marked expression of interleukin (IL)‐6 (IL‐6) (green) and IL‐17 (red) was observed in ocular infiltrative cells in rats treated with saline or nonstealth nanosteroids. Rats treated with stealth nanosteroids showed marked reduction of ocular infiltrative cells. Bar, 50 µm. (Reprinted with permission from Ref 69. Copyright 2011 Association for Research in Vision and Ophthalmology (Arvo))

Histopathologic features and scores of rats with experimental autoimmune uveoretinitis (EAU). Representative photographs taken 7 days after treatment with saline (a), 100 µg of nonstealth nanosteroids [poly(lactic acid) (PLA)] (b), and 100 µg of stealth nanosteroid [poly(lactic acid)–poly(ethylene glycol) (PLA–PEG)] (c) are shown. Note the disruption of the inner and outer segments in all areas for saline‐treated rats (a) and the preservation of structural integrity with stealth nanosteroids (c). White arrows: retinal folds and small granuloma formation; black arrows: inflammatory cellular infiltrates in the vitreous (original magnification, ×100). The severity of EAU in rats treated with saline, nonstealth nanosteroids, or stealth nanosteroids was graded 7 days after treatment. The scores for rats treated with stealth nanosteroids (1.5 ± 0.5) were significantly lower than for rats treated with saline (5.5 ± 0.8, P < 0.01) and nonstealth nanosteroids (3.0 ± 0.9, P < 0.05) (d). Data are shown as the mean ± SD (n = 6 in each group). (Reprinted with permission from Ref 69. Copyright 2011 Association for Research in Vision and Ophthalmology (Arvo))

Retinal vascular lesions in the Vldlr−/− retinas require continual production of excess reactive oxygen species (ROS). Vldlr−/− mice were injected at P28 with saline or nanoceria and killed 1 week later on P35. Analysis of VEGF levels by Western blots (a) showed a fourfold reduction (b) within 1 week of nanoceria injection. The numbers of IRN blebs (c) and SRN tufts (d) were also dramatically reduced. *P = 0.05; **P = 0.01. (Reprinted with permission from Ref 41. Copyright under Creative Commons Attributions License 2011)

Nanoceria inhibit the development of pathologic intra‐ and subretinal vascular lesions in the Vldlr−/− retina. Photomicrographs of whole mount retinas (a–c) and eyecups [retinal pigment epithelium (RPE), choroid, and sclera] (d–f) from P28 animals are shown. All retinal blood vessels were labeled green by the vascular filling assay. Wild type (WT) retinas (a) showed the normal web‐like retinal vasculature, whereas those from the Vldlr−/− mice (b) showed numerous intraretinal vascular lesions or ‘blebs’ (IRN blebs). See white arrows for examples. A single injection of nanoceria at P7 inhibited (c) the appearance of these lesions. Eyecups from WT mice (d) showed no subretinal neovascular (SRN) ‘tufts’ but those from Vldlr−/− mice (e) had many bright SRN tufts. A single injection of nanoceria on P7 inhibited the appearance of these SRN tufts (f). (Reprinted with permission from Ref 41. Copyright under Creative Commons Attributions License 2011)

Nanoceria reduce oxidative stress in the Vldlr−/− retina. Retinal sections from saline injected WT mice (a, d, g, j); saline injected Vldlr−/− mice (b, e, h, k) and CeO₂ injected (c, f, i, l) Vldlr−/− mice are shown as imaged by confocal microscopy. The 2′,7′‐dicholoro‐dihydro‐ fluorescein‐diacetate (DCF) assay (a–c) visualizes reactive oxygen species (ROS) as punctuate fluorescence and demonstrates a very low level of ROS in the normal (a), a considerable amount in the Vldlr−/− (b), and a greatly reduced amount in the retina of the Vldlr−/− mice injected with CeO₂ (c). Similar results were obtained with the other three assays. NADPH‐oxidase, (P47‐phox; d, e, f) a major producer of ROS, was very high in the Vldlr‐/− retina and almost reduced to control levels in the CeO₂ injected mice. Nitrotyrosine, (g, h, i) a reflection of oxidative activity due to increases in nitric oxide concentration, was highest in the Vldlr−/− retina and significantly reduced in the nanoceria injected mice. ROS‐mediated damage to DNA was indicated by the labeling of the retina with an antibody against a DNA adduct, 8‐hydroxy‐29‐deoxyguanosine (8‐OHdG; j, k, ) which showed little labeling in the control, significant labeling in the saline injected Vldlr−/− retina, and a greatly reduced amount in the nanoceria treated retina. DAPI (blue) was used to visualize the nuclei. (Reprinted with permission from Ref 41. Copyright under Creative Commons Attributions License 2011)

Bionic retina location within a patient. (a) The cable from the implanted chip in the eye leads under the temporal muscle to the exit behind the ear and connects with a wirelessly operated power control unit. (b) Position of the implant under the transparent retina. (c) Microphotodiode array (MPDA) photodiodes, amplifiers, and electrodes in relation to retinal neurons and pigment epithelium. (d) Patient with wireless control unit attached to a neckband. (e) Route of the polyimide foil (red) and cable (green) in the orbit in a three‐dimensional reconstruction of CT scans. (f) Photograph of the subretinal implant's tip at the posterior eye pole through a patient's pupil. (Reprinted with permission from Ref 191. Copyright 2011 The Royal Society of London)

Subretinal bionic retina. Some prostheses are placed on the retina (in direct contact with retinal ganglion cells); others are placed in the suprachoroidal space (in contact with the choroidal vasculature), and this implant is inserted into the subretinal space as illustrated here. (a) The microphotodiode array (MPDA) is a light sensitive 3.0 × 3.1 mm CMOS‐chip with 1500 pixel‐generating elements on a 20‐µm thick polyimide foil carrying an additional test field with 16 electrodes for direct electrical stimulation (DS test field). (b) The foil exits approximately 25 mm away from the tip at the equator of the eyeball and is attached to the sclera by means of a small fixation pad looping through the orbit to a subcutaneous silicone cable that connects through a plug behind the ear to a power control unit. (c) Magnification of the DS electrode array showing the 16 quadruple electrodes and their dimensions. (d) Pattern stimulation via DS array (e.g., ‘U’). (e, f) switching from a triangle to a square by shifting stimulation of a single electrode. (g) Magnification of 4 of the 1500 elements (‘pixels’), showing the rectangular photodiodes above each squared electrode and its contact hole that connects it to the amplifier circuit (overlaid sketch). (Reprinted with permission from Ref 191. Copyright 2011 The Royal Society of London)

Correlation of ankyrin_G‐hChR2 and PSD95‐eNpHR localization and function using immunostaining and electrophysiology. (a) Endogenous ankyrin_G‐Cy5 (magenta) in flat‐mount rabbit retina shown in the initial axon segment (arrowhead, inset) of ganglion cells. (b) Merge of ankyrin_G‐Cy5 and transfected ankyrin_G‐hChR2‐mCherry (red). Ankyrin_GhChR2 is localized to the soma and proximal dendrites. Colocalization of endogenous ankyrin_G (arrowhead) and mCherry is not apparent. (c) Cotransfection of untargeted enhanced green fluorescence protein (eGFP) (green) shows the complete cellular morphology (including axon, arrows). Scale bar represents 50 µm. (d) Endogenous PSD95‐Cy5 (magenta) is present in ganglion cell somata and dendritic terminals (arrowheads, inset). (e) Merge of PSD95‐Cy5 and transfected PSD95‐eNpHR‐eGFP (green). eNpHR‐eGFP is observed to colocalize with endogenous PSD95 in dendrites. (f) Cotransfection of untargeted mCherry (red) shows complete dendritic morphology of cell. Scale bar represents 50 µm. (g) Illumination of ankyrin_G‐hChR2‐mCherry (yellow) in ganglion cell soma with 50‐µm blue spot (10 mW/mm2) elicits robust spiking. Untargeted eGFP (green) was cotransfected to show complete morphology. Extracellular spike recordings from whole‐mount rabbit retina in the presence of l‐AP4 (20 µM), CPP (10 µM), and CNQX (10 µM) cocktail designed to block all photoreceptor‐driven synaptic transmission to ganglion cells. (h) Blue annulus (300 µm OD, 50 µm ID) covering only the cell dendrites and partial axon fails to elicit spiking. (i) A blue rectangular stimulus (200 × 900 µm) covering the entire axon also fails to elicit spiking. (j) Illumination of soma in ganglion cell expressing PSD95‐eNpHR‐eGFP (yellow) with 50‐µm yellow spot (10 mW/mm2) fails to silence spontaneous spiking. Untargeted mCherry (red) was cotransfected to show complete morphology. (k) Yellow annulus (300 µm OD, 50 µm ID) covering only the cell dendrites and partial axon effectively silences spikes. (l) Yellow rectangular stimulus (100 × 500 µm) covering the entire axon fails to silence spiking. (Reprinted with permission from Ref 183. Copyright 2011 Elsevier)

Humanized ChR2 (hChR2) and enhanced HaloR (eNpHR) construct schematics and differential transgene expression in ganglion cell soma and dendrites of whole‐mount rabbit retina. The calcium/calmodulin‐ dependent protein kinase II (CaMKIIa) promoter and woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) to drive high transgene expression levels in ganglion cells were used in all constructs. (a) Schematic of untargeted hChR2‐mCherry fusion. (b) Untargeted eNpHR‐eGFP fusion. (c) Postsynaptic density 95 (PSD‐95) targeting motif fused with hChR2‐mCherry for dendritic localization. (d) Ankyrin_G motif fused with eNpHR‐eGFP for somatic localization. (e) Ankyrin_G motif fused with hChR2‐mCherry. (f) PSD‐95 fused with eNpHR‐eGFP. (g) Confocal image of rabbit ganglion cell expressing ankyrin_G‐hChR2‐mCherry localized to the soma and proximal dendrites (red). (h) Same cell as (g) showing PSD95‐eNpHR‐eGFP localized primarily to the dendrites (green). (i) Merge of (g) and (h). Scale bar represents 100 µm. (j) PSD95‐hChR2‐mCherry localized to the dendrites. (k) Ankyrin_G‐eNpHR‐eGFP localized to the soma and proximal dendrites. (l) Merge of (j) and (k). Scale bar represents 100 µm. (m) Untargeted hChR2‐mCherry is localized throughout the plasma membrane. (n) Untargeted eNpHR‐eGFP is localized throughout the plasma membrane. (o) Merge of (m) and (n). Scale bar represents 100 µm. (Reprinted with permission from Ref 183. Copyright 2011 Elsevier)

Effect of structured surface on retinal progenitor cell behavior. (a) Attachment of retinal progenitor cells (RPCs) to substrate surfaces after 2 days growth. Substrate microtopography of 25 µm well PCL leads to significantly more RPC attachment compared to unstructured PCL and tissue culture polystyrene surfaces (TCPS). Fluorescence images of DAPI‐stained RPC nuclei attached to (b) TCPS, (c) unstructured PCL, and (d) 25‐µm well PCL. *P < 0.05, Student–Newman, Keuls test. Error bars indicate SD over three independent experiments. (Reprinted with permission from Ref 150. Copyright 2010 Springer)

Microfabrication of poly(ε‐caprolactone) (PCL) thin film with photolithography and soft lithography. (a) Schematic of PCL thin film scaffold fabrication. SU‐8 photoresist is spin‐cast onto a silicon wafer and exposed to ultraviolet light through a negative mask. Unexposed areas are not crosslinked and developed away, and polydimethylsiloxane (PDMS) is cured on the wafer. After peeling the PDMS mold from the wafer, PCL is spin‐cast on the mold and peeled from the surface. (b) A scanning electron micrograph of a PCL thin film with 25‐µm diameter wells. (c) Profile of PCL thin film. (Reprinted with permission from Ref 150. Copyright 2010 Springer)

Fluorescence microscopic evaluation of enhanced green fluorescent protein (EGFP) expression in transverse sections of retinal tissue 2 weeks after intravitreal injection. Immunostaining for EGFP in sections of the retina after delivery of (a) wild‐type self‐complementary adeno‐associated virus 2 (WT scAAV2), (b) serotype 2 tyrosine‐mutant Y444F, and (c) serotype 2 tyrosine‐mutant Y730F. Note intense EGFP staining throughout all retinal layers with Y444F mutant and predominant EGFP staining in the ganglion cell layer (GCL) with WT‐2 and Y730F. Calibration bar 100 µm. gcl, ganglion cell layer; ipl, inner plexiform layer; inl, inner nuclear layer; onl, outer nuclear; os, outer segment. (Reprinted with permission from Ref 131. Copyright 2009 Nature Publishing Group)