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. 2015:1271:391-410.
doi: 10.1007/978-1-4939-2330-4_25.

Gene therapy to rescue retinal degeneration caused by mutations in rhodopsin

Brian P Rossmiller  1 , Renee C Ryals, Alfred S Lewin
Affiliations

Gene therapy to rescue retinal degeneration caused by mutations in rhodopsin

Brian P Rossmiller et al. Methods Mol Biol. 2015.

Abstract (VSports最新版本)

Retinal gene therapy has proven safe and at least partially successful in clinical trials and in numerous animal models. Gene therapy requires characterization of the progression of the disease and understanding of its genetic cause. Testing gene therapies usually requires an animal model that recapitulates the key features of the human disease, though photoreceptors and cells of the retinal pigment epithelium produced from patient-derived stem cells may provide an alternative test system for retinal gene therapy. Gene therapy also requires a delivery system that introduces the therapeutic gene to the correct cell type and does not cause unintended damage to the tissue VSports手机版. Current systems being tested in the eye are nanoparticles, pseudotyped lentiviruses, and adeno-associated virus (AAV) of various serotypes. Here, we describe the techniques of AAV vector design as well as the in vivo and ex vivo tests necessary for assessing the efficacy of retinal gene therapy to treat retinal degeneration caused by mutations in the rhodopsin gene. .

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Figures (VSports)

Fig. 1
Fig. 1
Vector design and injection. (a) Typical AAV construct consisting of a promoter, intronic region, the gene of interest, and, optionally, a knockdown method. (b) Proper syringe location is at 45° angle to the cornea and inserted trans-scleral to deposit the virus subretinally
Fig. 2
Fig. 2
Optical coherence tomography. (a) Illustrated overview of the operational principle of spectral domain OCT. OCT builds an image of changes in refractive indexes and the depths of these changes in a sample using constructive and deconstructive interference of a broadband near infrared laser centered at 840 nm. The resulting intensity by frequency data is then converted using a Fourier transformation and graphed as intensity by time. Each point of data is called an A-scan. A linear cross section of the eye is built by compiling several A-scans to form a B-scan. (b) Proper placement of the mouse. Central animal tube rotated to 45° and base at 120° from laser. (c) Representation of the cell layers represented in an OCT B-scan. (d) The outer nuclear layer is measured using calipers to follow changes in thickness over time
Fig. 3
Fig. 3
Electroretinography. (a) Proper electrode placement with electrode centered and surrounding the cornea. (b) Different cell types each contribute different components of the recorded ERG. (c) Measured from the recorded ERG are the a-wave, b-wave, and the implicit time. The amplitude of the a-wave is measured from the baseline to the maximum negative deflection, while the amplityude of the b-wave is measured from the a-wave maximum to the peak of the b-wave in the positive direction.
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References

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