Scientists deploy CRISPR to preserve photoreceptors in mice
Silencing a gene called Nrl in mice prevents the loss of cells from degenerative diseases of the retina, according to a new study. The findings could lead to novel therapies for preventing vision loss from human diseases such as retinitis pigmentosa. The study was conducted by researchers at the National Eye Institute (NEI), part of the National Institutes of Health, and was published online today in Nature Communications.
The tissue in the back of the eye called the retina contains two types of cells that convert light into electrical signals sent to the brain. Rod photoreceptors enable vision in dim light, while cone photoreceptors enable color vision and the ability to see in well-lit conditions. Genetic mutations affect mostly rods, leading to night blindness such as that seen with retinitis pigmentosa. However, because rods also provide vital structural and nutritional support to cones, rod dysfunction or death can lead to cone degeneration and blindness.
In a series of studies, Anand Swaroop, Ph.D., chief of NEI's Neurodegeneration and Repair Laboratory, and his team asked if saving rods could prevent the loss of cones, thus preserving daylight and color vision.
During development, Nrl determines whether a photoreceptor precursor cell becomes a rod or a cone. In previous studies, Swaroop and colleagues showed that mice engineered to lack the Nrl gene develop cone-only retinas. Other researchers showed that if Nrl expression is knocked out in rods of mature mice, the cells gain cone-like features and survive despite rod gene mutations.
"The evidence suggested to us that coaxing rods into becoming more cone-like by knocking out Nrl was a potential strategy for overriding mutations that would otherwise lead to rod degeneration," said Swaroop. "Consequently, the neighboring cones would remain functional and viable."
To test the strategy, the researchers used a new genome editing technology called CRISPR, which stands for clustered regularly interspaced short palindromic repeats. CRISPR is like a molecular pair of scissors that can be programmed to snip a specific DNA sequence with precision.
NEI postdoctoral research fellow and the study's first author, Wenhan Yu, Ph.D., developed a method for applying CRISPR in photoreceptors. His strategy uses an adeno-associated virus (AAV) as a carrier, or vector, to introduce CRISPR into retinal cells. Yu tested this genome editing tool to remove the Nrl gene in wild-type mice and three different mouse models of retinal degeneration. By measuring gene expression and examining the retinal cells, the researchers confirmed that rods became more cone-like, as predicted. Although these cone-like rods could not detect light, they survived and improved survival of their neighboring cones.
In all three mouse models, rod degeneration was prevented or slowed, although less benefit was achieved when the therapy was introduced in older animals. Importantly, the benefit was evident in all three models, regardless of the specific gene defect in the mouse.
"Unlike conventional gene therapy, in which a normal gene is introduced to replace the defective gene, this approach could treat retinal degeneration caused by a variety of mutant genes," explained Zhijian Wu, Ph.D., head of the NEI Ocular Gene Therapy Core and senior author of the study.
More research is needed before the therapy is ready for testing in a clinical trial. The safety of CRISPR has yet to be established and information is needed about its possible adverse effects. Nevertheless, these findings provide proof of concept for CRISPR-based therapies for degenerative retinal diseases.
More information: Yu W, Mookherjee S, Chaitankar V, Hiriyanna S, Kim JW, Brooks M, Ataeijiannati Y, Sun X, Dong L, Li T, Swaroop A, Wu Z. 2017. Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice. Nature Communications.
Utafiti wa CRISPR unaonyesha kuwa kufuta jeni linaloitwa NrL katika panya huzuia kupoteza seli kutokana na magonjwa ya retina. Matokeo hayo yanaweza kuongoza kwenye matibabu mapya ya kuzuia upotezaji wa maono kutokana na magonjwa ya binadamu kama vile retinitis pigmentosa. Utafiti huo ulifanywa na watafiti wa Taasisi ya Kitaifa ya Jicho (NEI), sehemu ya Taasisi ya Kitaifa ya Afya, na ulichapishwa mtandaoni leo katika Nature Communications. Matumbo ya nyuma ya jicho huitwa retina na yana aina mbili za chembe zinazobadilisha mwanga kuwa ishara za umeme zinazopelekwa kwenye ubongo. Vipokezi vya rangi vya fimbo huwezesha kuona katika mwanga dhaifu, huku vipokezi vya rangi vya koni vikifanya iwezekane kuona rangi na uwezo wa kuona katika hali zenye mwangaza mzuri. Mabadiliko ya chembe za urithi huathiri hasa vijiti, na kusababisha upofu wa usiku kama ule unaoonekana na retinitis pigmentosa. Hata hivyo, kwa sababu vijiti pia hutoa msaada muhimu wa muundo na lishe kwa koni, kutofanya kazi vizuri kwa vijiti au kifo cha koni kunaweza kusababisha uharibifu wa koni na upofu. Katika utafiti uliofanywa na Anand Swarup, PhD, mkuu wa maabara ya Neurodegeneration na Urekebishaji wa NEI, na timu yake, walijibu swali hili: Je, vijiti vya kuokoa vinaweza kuzuia kupoteza cones, na hivyo kuhifadhi mwanga wa mchana na maono ya rangi? Wakati wa maendeleo, Nrl huamua kama seli ya mtangulizi wa photoreceptor inakuwa fimbo au koni. Katika utafiti wa awali, Swaroop na wenzake walionyesha kwamba panya waliobuniwa kutokuwa na jeni la Nrl huendeleza retina za koni tu. Watafiti wengine walionyesha kwamba ikiwa usemi wa Nrl unashindwa katika vijiti vya panya wakomavu, seli hupata sifa za koni na kuishi licha ya mabadiliko ya jeni la kijiti. "Ushahidi ulituonyesha kwamba kushawishi fimbo kuwa kama koni kwa kuondoa Nrl ilikuwa mkakati wa uwezekano wa kufuta mabadiliko ambayo vinginevyo yangesababisha uharibifu wa fimbo, ""Swaroop alisema." "Kwa hiyo, koni zilizo karibu zingebaki zikiwa na utendaji na zikiwa hai". Kwa mfano, utafiti wa CRISPR unaonyesha kwamba kuna aina ya chembe za urithi zinazojulikana kama clustered regularly interspaced short palindromic repeats. CRISPR ni kama safu ya molekuli ya scissors ambayo inaweza kupangwa kwa vipande vya DNA kwa usahihi. Mtafiti wa postdoctoral wa NIU na mwandishi wa kwanza wa utafiti huo, Wenhan Yu, Ph.D., aliendeleza njia ya kutumia CRISPR katika photoreceptors. "Kama ilivyoelezwa hapo juu, ""virusi vya adeno-associated (AAV) ni njia ya kuingiza CRISPR katika chembe za retina.""" Yu alijaribu chombo hiki cha kuhariri genome kuondoa jeni la Nrl katika panya wa aina ya mwituni na mifano mitatu tofauti ya panya ya degeneration ya retina. Kwa kupima maonyesho ya jeni na kuchunguza chembe za retina, watafiti walithibitisha kwamba vijiti vilikuwa na umbo la koni, kama ilivyotarajiwa. Ingawa fimbo hizo zenye umbo la koni hazikuweza kugundua nuru, ziliokoka na kuboresha uwezo wa kuokoka wa koni zilizo karibu nazo. Katika mifano yote mitatu ya panya, degeneration ya fimbo ilizuiliwa au kupunguzwa, ingawa faida kidogo ilifikiwa wakati tiba ilianzishwa kwa wanyama wakubwa. Jambo la muhimu ni kwamba faida hiyo ilionekana wazi katika mifano yote mitatu, bila kujali kasoro ya chembe za urithi katika panya. "Tofauti na tiba ya kawaida ya jeni, ambayo jeni ya kawaida huingizwa kuchukua nafasi ya jeni lisilofaa, mbinu hii inaweza kutibu degeneration ya retina inayosababishwa na jeni tofauti, ""alieleza Zhijian Wu, PhD, mkuu wa NEI Ocular Gene Therapy Core na mwandishi mwandamizi wa utafiti huo." Utafiti zaidi unahitajika kabla ya tiba hiyo kuwa tayari kwa ajili ya majaribio ya kliniki. Usalama wa CRISPR bado haujathibitishwa na habari inahitajika kuhusu athari zake mbaya zinazowezekana. Utafiti huo unaonyesha kuwa kuna uwezekano wa kugundua tiba ya CRISPR kwa magonjwa ya retina. Habari zaidi: Yu W, Mookherjee S, Chaitankar V, Hiriyanna S, Kim JW, Brooks M, Ataeijiannati Y, Sun X, Dong L, Li T, Swaroop A, Wu Z. Mwaka wa 2017. Uharibifu wa NRL na CRISPR-Cas9 inayotolewa na AAV huzuia degeneration ya retina katika panya. Mawasiliano ya Asili.