One of the greatest信明 barriers to treating neural de化年generation and neur農煙al tissue injury is t嗎現he fact that neurons do no裡明t proliferate, 得通and there are on拍習ly a few areas of the brain that exhi電熱bit neurogenesis. I去動t is possible to conve厭聽rt one somatic cell type to anoth討熱er with the best-known exampl雜懂e being the reprogramming of術化 fibroblasts to induced站路 pluripotent stem (iPS) c學火ells by “Yamanaka間但 factors” (1). More recently,筆小 direct transdifferen她內tiation of cells to neuro外算ns has been propo呢跳sed for treating a number of n得她eurodegenerative disorders (2). Spec男舞ifically targeting certain somatic 弟那cells circumvents immune recogn農綠ition and bypasses the need t畫身o revert back to an embryonic s那會tate thus avoiding the loss of es站喝sential age-related and epigenetic fac花謝tors. Glial cells南暗 represent a model target since they a要費re plentiful, proliferate in能店 response to injury and m好些ost importantly, are 美技highly plastic. However, w他風hile the conversion of gl從民ial cells to functional neurons黃老 represents an at海工tractive therapeutic approach 師如for a number of neurodegener件學ative disorders, t哥坐here has so far been 懂湖limited success (3).
Two recent papers爸計 from Qian et al (不笑4) and Zhou et al (5) dem兵自onstrate that reduced光車 expression of the RNA bindi哥間ng protein PTB (encoded b個如y Ptbp1-polypyrimidine tract-binding山中 protein) allows the direct conversion又農 of glial cells to neurons. PTB and 雜子it’s neuronal anal河間ogue nPTB are both d拍月own regulated during neurogenesis放門 (6) and in vitro knockdown of Ptb林劇p1 is sufficient for conversion 水飛of mouse fibroblasts to neuron雨靜s (7). Through regulation of a音什 microRNA circuit comprisi市下ng both positive and negative feedba坐睡ck loops, PTB inhibits the tran信得scriptional represso土的r REST that functio討西ns to suppress neuronal這司 gene targets.
In the work by Qian et al, conversi北要on of mouse astrocytes to 西購neurons was demonstrated by lentiviral章現-mediated shRNA knockdo做放wn (KD) of Ptbp1 草靜;(shPTB) resulting in neuronal 身女morphology and expressio票國n of neuronal ma靜舊rkers TUJ1 and MAP2 concomitant w通海ith suppression of astrocyte-spec資了ific genes. The respo志水nse of shPTB converted neurons to司如 synaptic input was al刀爸so confirmed by 照文patch clamp recordings. T都地o demonstrate in vivo rep作從rogramming of astrocytes, AA看動V expressing shPTB (together with a l機弟oxP-Stop-LoxP(LSL河數)-RFP) were injected into t湖場he substantia n現志igra of wild type and transgen章不eic mice expressing Cre dri們近ven by the astrocyte身腦-specific marker GFAB. Ove討少r time, the percentage of R靜高FP positive cells expressing matu數外re neuronal markers increased in AA小如V-shPTB injected mice thus demons生事trating astrocyte-to-neuro金離n conversion. Su個信bsequent experim紙都ents demonstrated sh服工PTB-targeted astrocytes are able to ma錯男ture into dopaminergi跳我c (DA) neurons offering 請上a potential resource to res老員tore dopamine levels in Park國上inson’s disease. Using an笑自 established model in which DA到會 neurons are ablated by 6-hydr制電oxydpamine (6-OH學老DA), depletion of stri去文atal dopamine leading to los跳讀s of neurons in多去 the substantia nigra was examined路大 following injecti生唱on with empty AAV or AAV-shPTB. In cont兵著rast to control treated mic慢資e, treatment of mice with AAV-shPT師懂B was able to restore the number of離大 neuronal cell bo司的dies together with a robust re線音storation of striatal dopamine 照坐and activity-induc廠她ed dopamine release. 門窗Although the 6-OHDA mouse model does no到靜t fully recapitul呢妹ate the phenotype of Parkinson’s,舊站 mice do exhibit motor phenotypes s了舊uch as contralateral forelimb們著 dysfunction. In young mi黃花ce at least, treatment with AAV-shPTB 吃們was able to correct three mo訊術tor phenotypes. Finally, to addres明他s clinical intervention, several antis對作ense oligonucleotides (ASOs) were西作 screened for the飛能ir ability to reduce紅雨 PTB in mouse astrocytes水家 and the top performing ASOs were t分開hen assessed for their abil月綠ity to restore motor function in the 6很文-OHDA model. ASOs were able t說了o convert astrocytes to f錢河unctional neurons and 睡筆importantly were abl費風e to rescue impa照事ired motor function.
In the paper by Zhou et al黃了, PTB depletion was北科 achieved by targeting with 快讀CRISPR-Cas13d (CasRx) which speci視化fically targets RNA (8區光). Following screening of several gRNAs有還 to edit and reduce答區 Ptbp1 mRNA levels, the ability 上黑of PTB to convert Müller glial (森議MG) cells to neuron們低s (Retinal ganglion cells – RGCs)好外 was examined. An AAV expressing dua空讀l gRNAs targeting Ptbp1 嗎通and CasRx driven by the astrocyte-關白specific promoter GFAP (AAV-G會上FAP-CasRx-Ptbp1) was injected資綠 into the eyes of Rosa-CAG房會-LSL-tdTomato mice tog刀票ether with AAV-GFAP-歌司Cre-GFP to identify MG cells. Conver聽離sion of MG cells to differe少遠nt subtypes of RGCs was confirmed by co爸來-staining of tdTomato posi頻費tive cells with several RGC markers s開麗uch as Brn3a and Rbpms. No些業 positive staining was obs銀火erved in retinas of mic司生e injected with control virus化火 (AAV-GFAP-CasRx-co頻都ntrol gRNAs).
To determine whether PTB deplet房美ed MG cells can replenish R文話GCs following injury, RGCs 上道were depleted by injection wi話個th N-methyl-D-aspa樂黑rtate (NMDA). When AAV-GFAP-小議CasRx-Ptbp1 was injected 2-3 w畫她eeks later, the number of MG-conve件看rted RGCs was significantly筆相 increased as was the response to lig間山ht stimulation. RGC projections to林海 the dorsal lateral geniculate個爸 nucleus (dLGN) and supe友算rior colliculus (SC) in the brain 話還are responsible for relaying妹爸 visual information. In NMDA/雪個AAV-GFAP-CasRx-Ptbp1 treated mice, t信那dTomato positve 空業axons were observed in the d道訊LGN and SC confirming the presence of作鐵 newly formed axons. These 了化MG-to-RGC derived projectio風厭ns partially re嗎風stored visual responses as determ也遠ined by visually evoked potentials 這坐(VEPs).
As with Qian et al, 一就Zhou et al next examined whether PTB d風去epleted astrocyt那刀es are capable of replenishing DA車可 neurons in the s道兒ubstantia nigra. AAV-GFAP-C又資asRx-Ptbp1 toget銀志her with AAV-GFA紅對P-mCherry were injected into開白 the striatum of 中日wild type mice. High林一 levels of CasRx expression was observe技子d with PTB reduction 站腦confirmed by imm慢下unostaining. Co-staining of m照鄉Cherry positive cells wit拍答h neuronal markers revealed conversio匠爸n of astrocytes to neuronal cells.工得 The 6-OHDA mouse model of 煙熱Parkinson’s was employed with AAV-如新GFAP-CasRx-Ptbp1 inje車年cted 3 weeks after 6-OHDA i明上nfusion. In contrast to co友線ntrol treated mice, a high percenta鄉資ge of cells from AAV-GFAP-Ca校了sRx-Ptbp1 injected mice expres農件sed mCherry and dopamine neuro影行n markers such a笑什s tyrosine hydroxylase (T朋購H) and Slc6a3 suggest能門ing derivation from astrocytes呢見. Converted DA neurons舊老 appeared to be able to release d有林opamine since mCherry positive cell車術s stained positive for vesicular友拍 monoamine transporter 2 (VMAT2), an es我冷sential protein requir計黃ed for dopamine release. Lastly, 行鐵to elucidate wh生好ether AAV-GFAP-Ca站照sRx-Ptbp1 transduced astrocyte習靜s could alleviate motor function校黑 symptoms in the 6-OHDA我亮 PD mouse model, contralateral rotation照視 and rotarod test風行s were performed. In both cases mic生雜e treated with AAV-GFAP-CasRx-Ptbp1 pe農小rformed better.
Since it was demonstrated that MyoD ca歌放n convert fibroblasts to myoblasts (他身9), several other tran玩都scription factors have been sho鐘那wn to induce transdi花黃fferentiation. 錯湖Additionally, powerful tools s做子uch as Mogrify (10) ex看是ist to predict transcription fa師用ctor “cocktails” fo愛件r reprogramming a p科店lethora of human cells. &土動nbsp;While additional studies are ne樹區eded to address longevity of converted 唱跳neurons, age-relat上睡ed restrictions, specificity a藍女nd adequate target時雪ing efficiency, the 懂校above work highligh靜雪ts a real potential for di現城rect in vivo glial-to音玩-neuronal fate swit民熱ching by suppression of a sing個票le RNA binding protein thus providin一離g a new approach for t開這herapeutic application for Parkin學務son’s disease a子購nd other neurological disorders制就.
VectorBuilderoffers second-generation recomb問舊inant AAV packaging for multiple森影 serotypes (1, 2, 3, 4, 5, 化房6, 6.2, 7, 8, 9不的, rh10, DJ, DJ/8, PHP.eB, PHP.S,吧費 AAV2-retro, AA鐵西V2-QuadYF and AAV2.7m8). We o大物ffer shRNA, single票能 and dual gRNA vectors and can package朋校 both ssAAV (single快月-stranded AAV) and scAAV (self計路-complementary AAV). Contact us to find out more, or for hel分訊p designing your custom vectors. 農費Use our award-winning online platform物話 to design and order事影 custom vectors草近 specific to your research needs.如答
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