Research to Prevent Blindness; Experimental assistance from Takayuki Nagasaki; This research complies with the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research. KJW is supported by NIH grants 5T32EY013933 and 5T32DK007647-20. VBM is supported by NIH grant K08EY020530.

1. Assemble Devices for the Subretinal Injection
<ol>
 <li>Purchase or make a 100 &mu;m diameter needle from a glass capillary tube. This can be done manually by using a Sutter P-97 pipet puller or other similar equipment. The end of the capillary tube will be heated and pulled until it reaches the desired diameter (100 &mu;m). A smaller diameter needle can be used for gene therapy vectors; however, this is the recommended diameter for cell injection without damage to the cells or the eye. Compared to steel needles, t...

<ol>
	<li>Abe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+the+retina+using+pigment+epithelial+cell+transplantation.">Regeneration of the retina using pigment epithelial cell transplantation.</a> Nihon Ganka Gakkai Zasshi. 106, 778-803 (2002).</li><li>Jacobson, S. G., Cideciyan, A. V., Ratnakaram, R., Heon, E., Schwartz, S. B., Roman, A. J., Peden, M. C., Aleman, T. S., Boye, S. L., Sumaroka, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+for+Leber+congenital+amaurosis+caused+by+RPE65+mutations:+safety+and+efficacy+in+15+children+and+adults+followed+up+to+3+years.">Gene therapy for Leber congenital amaurosis caused by RPE65 mutations: safety and efficacy in 15 children and adults followed up to 3 years.</a> Arch. Ophthalmol. 130, 9-24 (2011).</li><li>Schwartz, S. D., Hubschman, J. P., Heilwell, G., Franco-Cardenas, V., Pan, C. K., Ostrick, R. M., Mickunas, E., Gay, R., Klimanskaya, I., Lanza, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+trials+for+macular+degeneration:+a+preliminary+report.">Embryonic stem cell trials for macular degeneration: a preliminary report.</a> Lancet. 379, 713-720 (2012).</li><li>Wang, N. K., Tosi, J., Kasanuki, J. M., Chou, C. L., Kong, J., Parmalee, N., Wert, K. J., Allikmets, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+reprogrammed+embryonic+stem+cells+improves+visual+function+in+a+mouse+model+for+retinitis+pigmentosa.">Transplantation of reprogrammed embryonic stem cells improves visual function in a mouse model for retinitis pigmentosa.</a> Transplantation. 89, 911-919 (2010).</li><li>Song, B. J., Tsang, S. H., Lin, C. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+models+of+retinal+degeneration+and+targets+for+gene+therapy.">Genetic models of retinal degeneration and targets for gene therapy.</a> Gene Ther. Mol. Biol. 11, 229-262 (2007).</li><li>Tosi, J., Sancho-Pelluz, J., Davis, R. J., Hsu, C. W., Wolpert, K. V., Sengillo, J. D., Lin, C. S., Tsang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus-mediated+expression+of+cDNA+and+shRNA+slows+degeneration+in+retinitis+pigmentosa.">Lentivirus-mediated expression of cDNA and shRNA slows degeneration in retinitis pigmentosa.</a> Exp. Biol. Med. 236, 1211-1217 (2011).</li><li>Tucker, B. A., Park, I. H., Qi, S. D., Klassen, H. J., Jiang, C., Yao, J., Redenti, S., Daley, G. Q., Young, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+adult+mouse+iPS+cell-derived+photoreceptor+precursors+restores+retinal+structure+and+function+in+degenerative+mice.">Transplantation of adult mouse iPS cell-derived photoreceptor precursors restores retinal structure and function in degenerative mice.</a> PLoS One. 6, e189992 (2011).</li><li>Mahajan, V. B., Mondino, B. J., Tsang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A high-throughput Mouse Eye Phenomics System. , (2010).</li><li>Chang, B., Hawes, N. L., Hurd, R. E., Davisson, M. T., Nusinowitz, S., Heckenlively, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+degeneration+mutants+in+the+mouse.">Retinal degeneration mutants in the mouse.</a> Vision Res. 42, 517-525 (2002).</li><li>Hawes, N. L., Smith, R. S., Chang, B., Davisson, M., Heckenlively, J. R., John, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+fundus+photography+and+angiography:+a+catalogue+of+normal+and+mutant+phenotypes.">Mouse fundus photography and angiography: a catalogue of normal and mutant phenotypes.</a> Mol. Vis. 5, 22 (1999).</li><li>Won, J., Shi, L. Y., Hicks, W., Wang, J., Hurd, R., Naggert, J. K., Chang, B., Nishina, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+resources+for+vision+research.">Mouse model resources for vision research.</a> J. Ophthalmol. 2011, 391384 (2011).</li></ol>

This video technique provides instructions on completing the subretinal injection surgical procedure successfully, and ensuring that the gene therapy vector or stem cells are placed in the location necessary to efficiently treat the ophthalmic disease. This technique allows for the targeting of retinal cells such as the RPE or photoreceptors, since it places the gene therapy vectors or stem cell-derived tissues in the vicinity of these cells. Previous methods involved intravitreal injections, where the fluid is placed wi...

<table border="1">
<tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>0.8-1.10 x 100 mm Capillary Tube (glass)</td>
 <td>Kimble Glass, Inc.</td>
 <td>34502 99</td>
 <td></td>
 </tr>
 <tr>
 <td>Flaming/Brown Micropipette Puller </td>
 <td>Sutter Instrument</td>
 <td>P-97</td>
 <td>Narishige microforge can be used instead.&nbsp; Catalog #MF-900 </td>
 </tr>
 <tr>
 <td>Sigmacote</td>
 <td>Sigma Aldrich</td>
 <td>SL2-25ML</td>
 <td>Silicone</td>
 </tr>
 <tr>
 <td>Dubecco's Phosphate Buffered Saline with Calcium Chloride and Magnesium Chloride</td>
 <td>Gibco-Invitrogen</td>
 <td>14040-133</td>
 <td></td>
 </tr>
 <tr>
 <td>Safety-Lok 25 3/4G x 12"; Blood Collection Set</td>
 <td>B-D Vacutainer</td>
 <td>367298</td>
 <td></td>
 </tr>
 <tr>
 <td>1 ml Sub-Q 26 5/8G Slip-Tip Syringe</td>
 <td>Becton-Dickinson</td>
 <td>309597</td>
 <td></td>
 </tr>
 <tr>
 <td>0.5-10 &mu;l Finnpipette II Adjustable-Volume Pipetter</td>
 <td>Fisherbrand</td>
 <td>21-377-815</td>
 <td></td>
 </tr>
 <tr>
 <td>1-200 &mu;l Natural Beveled Tips</td>
 <td>USA Scientific, Inc.</td>
 <td>1111-1700</td>
 <td></td>
 </tr>
 <tr>
 <td>Discovery.V8 Stereo Microscope</td>
 <td>Zeiss</td>
 <td>MC1500</td>
 <td></td>
 </tr>
 <tr>
 <td>60 mm x 15 mm Style Treated Polystyrene Cell Culture Dish</td>
 <td>Corning Incorporated</td>
 <td>430166</td>
 <td></td>
 </tr>
 <tr>
 <td>Vannas Straight Scissors</td>
 <td>Storz Ophthalmics</td>
 <td>E3383 S</td>
 <td></td>
 </tr>
 <tr>
 <td>Curved Dressing Forceps with Serrations Delicate</td>
 <td>Storz Ophthalmics</td>
 <td>E1408</td>
 <td></td>
 </tr>
 <tr>
 <td>15 Degree Microsurgery Knife</td>
 <td>Wilson Ophthalmic Corp.</td>
 <td>091204</td>
 <td></td>
 </tr>
 <tr>
 <td>Ketamine</td>
 <td>Ketaset III</td>
 <td>NADA #45-290</td>
 <td></td>
 </tr>
 <tr>
 <td>Xylazine</td>
 <td>Lloyd Laboratories</td>
 <td>NADA #139-236</td>
 <td></td>
 </tr>
 <tr>
 <td>Bupivacaine (Marcaine)</td>
 <td>AstraZeneca</td>
 <td>N/A</td>
 <td></td>
 </tr>
 <tr>
 <td>Buprenorphine </td>
 <td>Sigma Aldrich</td>
 <td>B9275</td>
 <td></td>
 </tr>
 </tbody>
</table>

subretinal injection of gene therapy vectors and stem cells in the perinatal mouse eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

A drawing of the mouse eye is shown with major structures labeled for reference, with arrows displaying the locations for both intravitreal and subretinal injection surgical procedures (arrowheads, Figure 1). Gene therapy vectors, such as the lacZ lentiviral vector (Figure 2), can be injected using these locations. Additionally, stem cells, such as mouse embryonic stem cells (Figure 3), can also be transplanted at these sites in the mouse eye. 
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Watch this Scientific Journal Video about Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye at JoVE.com

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

subretinal-injection-gene-therapy-vectors-stem-cells-perinatal-mouse

Research

JoVE Journal

Medicine

32.8K visualizações.  Columbia University. Esta técnica cirúrgica ilustra a injecção de vectores de terapia genética e de células estaminais para o espaço sub-retiniano do olho do rato.