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Cited by 109Retinal Development and DisordersRNA Interference and Gene DeliveryAdvanced Drug Delivery Systems

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A Partial Structural and Functional Rescue of a Retinitis Pigmentosa Model with Compacted DNA Nanoparticles

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Cai, Xue, Nash, Zack, Conley, Shannon M., Fliesler, Steven J., Cooper, Mark J., Naash, Muna I.·2009·PLoS ONE
DOI 10.1371/journal.pone.0005290PMC PMC2669177
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Abstract

Previously we have shown that compacted DNA nanoparticles can drive high levels of transgene expression after subretinal injection in the mouse eye. Here we delivered compacted DNA nanoparticles containing a therapeutic gene to the retinas of a mouse model of retinitis pigmentosa. Nanoparticles containing the wild-type retinal degeneration slow (Rds) gene were injected into the subretinal space of rds(+/-) mice on postnatal day 5. Gene expression was sustained for up to four months at levels up to four times higher than in controls injected with saline or naked DNA. The nanoparticles were taken up into virtually all photoreceptors and mediated significant structural and biochemical rescue of the disease without histological or functional evidence of toxicity. Electroretinogram recordings showed that nanoparticle-mediated gene transfer restored cone function to a near-normal level in contrast to transfer of naked plasmid DNA. Rod function was also improved. These findings demonstrate that compacted DNA nanoparticles represent a viable option for development of gene-based interventions for ocular diseases and obviate major barriers commonly encountered with non-viral based therapies.

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Figures

Figure 1 Injection of NMP nanoparticles into P5 rds +/− animals increases Rds mRNA levels. cDNA from eyes injected with saline, naked DNA (A) or nanoparticle DNA (B) at PI-2 through PI-120 was prepared and analyzed by qRT-PCR to determine relative Rds mRNA levels. Because Rds primers amplify from the NMP (nanoparticle) and the WT (endogenous) allele but not from the Rds mutant allele, expression values are reported as fold change from the uninjected contralateral control eye. Values shown are averages±S.D. (N = 3–6 mice per group). (A) Injection of saline or naked DNA does not alter Rds message levels at any time point. (B) Conversely, injection of both CBA-NMP and IRBP-NMP compacted DNA nanoparticles leads to a significant, two- to four-fold increase in total Rds message level compared to the naked DNA injected eyes (*p<0.001, **p<0.05. This increase persists through the last time point examined (PI-120).
Figure 1 Injection of NMP nanoparticles into P5 rds +/− animals increases Rds mRNA levels. cDNA from eyes injected with saline, naked DNA (A) or nanoparticle DNA (B) at PI-2 through PI-120 was prepared and analyzed by qRT-PCR to determine relative Rds mRNA levels. Because Rds primers amplify from the NMP (nanoparticle) and the WT (endogenous) allele but not from the Rds mutant allele, expression values are reported as fold change from the uninjected contralateral control eye. Values shown are averages±S.D. (N = 3–6 mice per group). (A) Injection of saline or naked DNA does not alter Rds message levels at any time point. (B) Conversely, injection of both CBA-NMP and IRBP-NMP compacted DNA nanoparticles leads to a significant, two- to four-fold increase in total Rds message level compared to the naked DNA injected eyes (*p<0.001, **p<0.05. This increase persists through the last time point examined (PI-120).CC BY · PubMed Central
Figure 2 Transferred NMP co-localizes with endogenous RDS. Frozen retinal sections from eyes collected at multiple ages (PI-2 to PI-30) were immunostained for NMP (mAB 3B6, green) and total RDS (RDS-CT, red) with a nuclear counterstain (DAPI, blue). Transferred RDS from eyes injected with CBA-NMP (A) and IRBP-NMP (B) nanoparticles is detected at PI-2. Expression remains strong through the latest time point analyzed (PI-30) and co-localizes with native RDS. Expression is limited to the OSs or nascent OSs and is not detected in any other retinal cell types, subcellular compartments or layers. (C) No NMP is detected in saline-injected control eyes, but native RDS is detected beginning at PI-2 (P7), consistent with normal ocular development. Scale bars, 20 µm. N = 3–5 mice per group. Abbreviations: RPE, retinal pigment epithelium; OS, outer segment layer; ONL, outer nuclear layer; INL, inner nuclear layer.
Figure 2 Transferred NMP co-localizes with endogenous RDS. Frozen retinal sections from eyes collected at multiple ages (PI-2 to PI-30) were immunostained for NMP (mAB 3B6, green) and total RDS (RDS-CT, red) with a nuclear counterstain (DAPI, blue). Transferred RDS from eyes injected with CBA-NMP (A) and IRBP-NMP (B) nanoparticles is detected at PI-2. Expression remains strong through the latest time point analyzed (PI-30) and co-localizes with native RDS. Expression is limited to the OSs or nascent OSs and is not detected in any other retinal cell types, subcellular compartments or layers. (C) No NMP is detected in saline-injected control eyes, but native RDS is detected beginning at PI-2 (P7), consistent with normal ocular development. Scale bars, 20 µm. N = 3–5 mice per group. Abbreviations: RPE, retinal pigment epithelium; OS, outer segment layer; ONL, outer nuclear layer; INL, inner nuclear layer.CC BY · PubMed Central
Figure 3 Transferred NMP leads to increased expression of photoreceptor-specific proteins in the rds +/− retina. (A–C) cDNA was collected at PI-30, and message levels of photoreceptor genes were analyzed by qRT-PCR. (A) CBA-NMP nanoparticle injection leads to a modest increase in Rom-1 message levels, while IRBP-NMP nanoparticle injection increases expression four- to five-fold over levels in uninjected control eyes. (B,C) CBA-NMP and IRBP-NMP nanoparticle injections lead to increases in rod (B) and cone (C) opsins. (A–C); N = 3 animals per group. (D–F) Protein levels at PI-30 after nanoparticle injection were examined. Representative SDS-PAGE/Western blots from individual retinas are shown (N = 5–6 animals per group). (D) CBA-NMP and IRBP-NMP nanoparticle injections increase RDS and ROM-1 protein levels (protein load: 20 µg per lane). (E) Increases in rhodopsin protein (RHO) are detected after injection of both CBA-NMP and IRBP-NMP nanoparticles (protein load: 10 µg per lane). (F) No change in S-opsin (S-ops) protein level is detected after nanoparticle injection (protein load: 50 µg per lane). (G) Double immunolabeling for transferred RDS (mAB 3B6, green) and cone OSs (S-opsin, red) with nuclear counterstain (DAPI, blue) was performed on frozen sections from PI-30 eyes. Representative cones from two different animals are shown for each treatment. Cones in eyes injected with CBA-NMP or IRBP-NMP nanoparticles express transferred NMP (top and middle rows). Saline injected eyes express no transferred NMP (bottom row). Scale bar, 5 µm; N = 3–5 animals per treatment group. Abbreviations: OS, outer segment layer; IS, inner segment layer; ONL, outer nuclear layer * = p<0.05.
Figure 3 Transferred NMP leads to increased expression of photoreceptor-specific proteins in the rds +/− retina. (A–C) cDNA was collected at PI-30, and message levels of photoreceptor genes were analyzed by qRT-PCR. (A) CBA-NMP nanoparticle injection leads to a modest increase in Rom-1 message levels, while IRBP-NMP nanoparticle injection increases expression four- to five-fold over levels in uninjected control eyes. (B,C) CBA-NMP and IRBP-NMP nanoparticle injections lead to increases in rod (B) and cone (C) opsins. (A–C); N = 3 animals per group. (D–F) Protein levels at PI-30 after nanoparticle injection were examined. Representative SDS-PAGE/Western blots from individual retinas are shown (N = 5–6 animals per group). (D) CBA-NMP and IRBP-NMP nanoparticle injections increase RDS and ROM-1 protein levels (protein load: 20 µg per lane). (E) Increases in rhodopsin protein (RHO) are detected after injection of both CBA-NMP and IRBP-NMP nanoparticles (protein load: 10 µg per lane). (F) No change in S-opsin (S-ops) protein level is detected after nanoparticle injection (protein load: 50 µg per lane). (G) Double immunolabeling for transferred RDS (mAB 3B6, green) and cone OSs (S-opsin, red) with nuclear counterstain (DAPI, blue) was performed on frozen sections from PI-30 eyes. Representative cones from two different animals are shown for each treatment. Cones in eyes injected with CBA-NMP or IRBP-NMP nanoparticles express transferred NMP (top and middle rows). Saline injected eyes express no transferred NMP (bottom row). Scale bar, 5 µm; N = 3–5 animals per treatment group. Abbreviations: OS, outer segment layer; IS, inner segment layer; ONL, outer nuclear layer * = p<0.05.CC BY · PubMed Central
Figure 4 Expression of transferred NMP leads to partial functional rescue of the rds +/−phenotype. A subset of individual injected animals identified from PI-30 ERG analysis (Table 1) was chosen for follow-up. (A,B) Top: scotopic traces from naked DNA (gray) and nanoparticle (black) injected eyes at PI-30. Bottom: (A) Scotopic a-wave amplitudes from eyes injected with CBA-NMP nanoparticles are elevated at PI-30, but drop almost back to baseline at PI-60 and PI-120. (B) IRBP-NMP nanoparticle-injected animals retain improved rod function (as measured by scotopic a-wave) through PI-120. (C,D) Top: photopic traces from naked DNA (gray) and nanoparticle (black) injected eyes at PI-30. Bottom: (C) Cone function (as measured by photopic b-wave) does not remain substantially improved past PI-30 in eyes injected with CBA-NMP nanoparticles. (D) Photopic b-wave amplitudes in IRBP-NMP nanoparticle-injected animals are improved at PI-30 and continue improving at PI-60 (* = p<0.05, PI-30 vs. PI-60) before stabilizing at the last time point examined (PI-120). Amplitudes are means±standard error (N values are in Table 1).
Figure 4 Expression of transferred NMP leads to partial functional rescue of the rds +/−phenotype. A subset of individual injected animals identified from PI-30 ERG analysis (Table 1) was chosen for follow-up. (A,B) Top: scotopic traces from naked DNA (gray) and nanoparticle (black) injected eyes at PI-30. Bottom: (A) Scotopic a-wave amplitudes from eyes injected with CBA-NMP nanoparticles are elevated at PI-30, but drop almost back to baseline at PI-60 and PI-120. (B) IRBP-NMP nanoparticle-injected animals retain improved rod function (as measured by scotopic a-wave) through PI-120. (C,D) Top: photopic traces from naked DNA (gray) and nanoparticle (black) injected eyes at PI-30. Bottom: (C) Cone function (as measured by photopic b-wave) does not remain substantially improved past PI-30 in eyes injected with CBA-NMP nanoparticles. (D) Photopic b-wave amplitudes in IRBP-NMP nanoparticle-injected animals are improved at PI-30 and continue improving at PI-60 (* = p<0.05, PI-30 vs. PI-60) before stabilizing at the last time point examined (PI-120). Amplitudes are means±standard error (N values are in Table 1).CC BY · PubMed Central
Figure 5 Transferred NMP leads to structural rescue of the rds +/− phenotype. Light micrographs (top row) and electron micrographs (bottom row, N = 3–5 animals per group) from rds +/− were examined. (A) At PI-30, moderate ultrastructural rescue is detected in the OSs of nanoparticle injected eyes (arrows). (B) By PI-120 significant ultrastructural improvement in OSs of nanoparticle injected eyes is apparent. OS discs are properly aligned and flattened and OS do not exhibit the swirl-like structures typical of the rds +/−. RPE, retinal pigment epithelium; OS, outer segment layer; IS, inner segment layer; ONL, outer nuclear layer. Scale bar, 10 µm.
Figure 5 Transferred NMP leads to structural rescue of the rds +/− phenotype. Light micrographs (top row) and electron micrographs (bottom row, N = 3–5 animals per group) from rds +/− were examined. (A) At PI-30, moderate ultrastructural rescue is detected in the OSs of nanoparticle injected eyes (arrows). (B) By PI-120 significant ultrastructural improvement in OSs of nanoparticle injected eyes is apparent. OS discs are properly aligned and flattened and OS do not exhibit the swirl-like structures typical of the rds +/−. RPE, retinal pigment epithelium; OS, outer segment layer; IS, inner segment layer; ONL, outer nuclear layer. Scale bar, 10 µm.CC BY · PubMed Central

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  1. MJ Cooper, 2007, Nonviral gene transfer in the lung: from animal to human., Molecular therapy, S15, 315

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Metadata

Authors
Cai, X., Nash, Z., Conley, S. M., Fliesler, S. J., Cooper, M. J., & Naash, M. I.
Affiliations
  • University of Oklahoma Health Sciences Center (US)
  • Saint Louis University (US)
  • Copernicus Therapeutics (United States) (US)
Corresponding author
Naash, Muna I.
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2009-04-24
Volume
4
Issue
4
Pages
e5290
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0005290
PMC
PMC2669177
OpenAlex
W1969461625
Cited by
109
References
49
Citations by year
2026: 2 · 2025: 8 · 2024: 3 · 2023: 3 · 2022: 5 · 2021: 5 · 2020: 5 · 2019: 1 · 2018: 7 · 2017: 4 · 2016: 4 · 2015: 7 · 2014: 8 · 2013: 8 · 2012: 14
FWCI
5.24
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • Foundation Fighting Blindness
  • Research to Prevent Blindness
  • Oklahoma Center for the Advancement of Science and Technology
  • National Institutes of Health (EY018656)
  • National Eye Institute (R56EY010609, EY007361, EY10609, R01EY007361, R01EY018656, R01EY010609, EY018656)
Topics
  • Health Sciences › Medicine › Ophthalmology › Retinal Development and Disorders
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmaceutical Science › Advanced Drug Delivery Systems
Keywords
retinitis pigmentosa, gene therapy, subretinal injection, photoreceptors, cone function, rod function, electroretinogram, non-viral gene delivery, ocular gene therapy, mouse model
MeSH terms
Animals, Disease Models, Animal, DNA, Electroretinography, Immunohistochemistry, Intermediate Filament Proteins, Membrane Glycoproteins, Mice, Transgenic, Nerve Tissue Proteins, Retinitis Pigmentosa, Transgenes, Photoreceptor Cells, Vertebrate, Mice, Nanoparticles, Peripherins

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