Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: Implications for the therapeutic development of RNAi

Jodi L. McBride*, Ryan L. Boudreau*, Scott Q. Harper*†, Patrick D. Staber*, Alex Mas Monteys*, Ineˆ s Martins*, Brian L. Gilmore*, Haim Burstein‡, Richard W. Peluso‡, Barry Polisky§, Barrie J. Carter‡, and Beverly L. Davidson*¶ʈ**

Departments of *Internal Medicine, ¶Molecular Physiology and Biophysics, and ʈNeurology, University of Iowa, Iowa City, IA 52242; ‡Targeted Genetics, 1100 Olive Way, Suite 100, Seattle, WA 98101; and §Sirna Therapeutics, 1700 Owens Street, San Francisco, CA 94158

Communicated by David E. Housman, Massachusetts Institute of Technology, Cambridge, MA, February 27, 2008 (received for review February 10, 2008) Huntington’s disease (HD) is a fatal, dominant neurodegenerative in the setting of two normal mouse HD homolog (HDh) alleles. disease caused by a polyglutamine repeat expansion in exon 1 of Although allele-specific targeting of disease transcripts for HD the HD gene, which encodes the huntingtin . We and others therapy would be ideal, to date no prevalent SNP residing on the have shown that RNAi is a candidate therapy for HD because mutant transcript has been identified. Therefore, we undertook expression of inhibitory targeting mutant human HD trans- studies to identify inhibitory RNAs that would target both mouse genes improved neuropathology and behavioral deficits in HD HDh and human HD transcripts, with the intention of testing the mouse models. Here, we developed shRNAs targeting conserved efficacy of reducing the expression of both alleles in a knockin sequences in human HD and mouse HD homolog (HDh) mRNAs to model of HD (10). Here we describe the surprising finding of initiate preclinical testing in a knockin mouse model of HD. We neurotoxicity in mouse brain caused by some, but not all, shRNA screened 35 shRNAs in vitro and subsequently narrowed our focus expression vectors screened in vivo and the notable reduction in to three candidates for in vivo testing. Unexpectedly, two active toxicity after moving those toxic inhibitory RNAs into miRNA- shRNAs induced significant neurotoxicity in mouse striatum, al- based delivery systems. though HDh mRNA expression was reduced to similar levels by all three. Additionally, a control shRNA containing mismatches also Results induced toxicity, although it did not reduce HDh mRNA expression. shRNAs Cause Striatal Toxicity in Mice. We first designed and Interestingly, the toxic shRNAs generated higher antisense RNA screened shRNAs (driven by the mouse U6 promoter) that target levels, compared with the nontoxic shRNA. These results demon- conserved sequences spanning human HD and mouse HDh strate that the robust levels of antisense RNAs emerging from mRNAs [Fig. 1A and supporting information (SI) Table S1], shRNA expression systems can be problematic in the mouse brain. taking into consideration the most recent siRNA design rules Importantly, when sequences that were toxic in the context of (11–13). Silencing of HD mRNA measured by quantitative shRNAs were placed into artificial microRNA (miRNA) expression real-time PCR (QPCR) and dot blot analysis revealed a decrease systems, molecular and neuropathological readouts of neurotox- in huntingtin protein expression after transfection of shRNA icity were significantly attenuated without compromising mouse expression into mouse C2C12 and human-derived HEK HDh silencing efficacy. Thus, miRNA-based approaches may pro- 293 cell lines (data not shown). Of the 35 shRNAs tested, three vide more appropriate biological tools for expressing inhibitory were chosen for further study based on silencing efficacy. The RNAs in the brain, the implications of which are crucial to the shRNAs target sequences in exons 2, 8, and 30 of HD mRNAs development of RNAi for both basic biological and therapeutic and are henceforth referred to as sh2.4, sh8.2, and sh30.1, applications. respectively (Fig. 1B). Western blot analysis demonstrated that these shRNAs, but not mismatch (mis) control shRNAs, reduce gene therapy ͉ Huntington’s disease ͉ RNAi ͉ AAV endogenous huntingtin protein expression in mouse C2C12 cells (Fig. 1C). Similar results were seen in human-derived HEK 293 he ability of siRNAs to silence target genes was first demon- cells. Tstrated in 1998 by Andrew Fire et al. (1) and has since emerged To examine the long-term effects of brain-delivered shRNAs in as a revolutionary strategy to reduce target . RNAi the CAG140 knockin mouse model of HD (10), U6-shRNA occurs naturally in cells as a posttranscriptional regulatory mech- expression cassettes were cloned into adeno-associated viral vectors anism mediated by endogenous miRNAs (2–5). RNAi is hypoth- (AAV serotype 2/1) (Fig. 2A). AAVs also contained a humanized esized to have evolved as a cellular coping mechanism providing the Renilla GFP (hrGFP) expression cassette to identify the distribu- cell a means to decrease the expression of various deleterious tion and types of cells transduced. Five-week-old CAG140 knockin viruses and transposons (6, 7). In recent years, scientists have mice were injected bilaterally into the striatum with AAVsh2.4- coopted this biological process to reduce the expression of target GFP, AAVsh8.2-GFP, AAVsh30.1-GFP, or AAV-GFP (viral con- mRNAs by using exogenously applied siRNAs, shRNAs, or artifi- trol) and killed 15 weeks later. Robust expression of GFP was cial miRNAs. Aside from the widespread basic biological applica- tions of RNAi, the ability to reduce gene expression marks a major Author contributions: J.L.M., R.L.B., and S.Q.H. contributed equally to this work; J.L.M., advance toward the development of disease therapies, particularly R.L.B., S.Q.H., B.P., B.J.C., and B.L.D. designed research; J.L.M., R.L.B., S.Q.H., P.D.S., A.M.M., for dominantly inherited disorders. I.M., B.L.G., H.B., and R.W.P. performed research; J.L.M., R.L.B., S.Q.H., and B.L.D. analyzed Among the dominant diseases that may benefit from RNAi- data; and J.L.M., R.L.B., S.Q.H., and B.L.D. wrote the paper. based therapies is Huntington’s disease (HD). Our laboratory (8) Conflict of interest statement: B.L.D. was a consultant for Sirna Therapeutics, Inc.. and others (9) have previously demonstrated that partial reduction †Present address: Center for Gene Therapy, Department of Pediatrics, Ohio State University, of mutant huntingtin expression by viral delivery of shRNAs is Columbus, OH 43205. efficacious in preventing the development of motor deficits and **To whom correspondence should be addressed. E-mail: [email protected]. neuropathology in transgenic mouse models of HD. In these This article contains supporting information online at www.pnas.org/cgi/content/full/ proof-of-principal studies, the therapeutic effect on disease pheno- 0801775105/DCSupplemental. type was studied by knocking down a mutant human HD transgene © 2008 by The National Academy of Sciences of the USA

5868–5873 ͉ PNAS ͉ April 15, 2008 ͉ vol. 105 ͉ no. 15 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0801775105 Downloaded by guest on September 27, 2021 AAVshRNA-GFP A A ITR ITR 1 2 8 30 U6shRNA TTTT CMV hrGFP polyA

B sh2.4 B Anterior U U 5‘ C C GACCGUGUGAAUCAUUGUCUA C CUGGCACACUUAGUAACAGAU U 3‘-UU A G C U striatum striatum striatum striatum sh8.2 U U 5‘ C C CAGCUUGUCCAGGUUUAUGAA C GUCGAACAGGUCCAAAUACUU U 3‘-UU A G striatumglobus pallidus globus pallidus globus pallidus C U C 1.0 sh30.1 U U 5‘ C C GGAUACCUGAAAUCCUGCUUU C 0.8 CCUAUGGACUUUAGGACGAAA U 3‘-UU A G C U 0.6 * * * C 0.4 0.2 Relative HDh mRNA Untreatedsh8.2 sh2.4mis sh30.1 mis sh8.2 missh2.4sh30.1shLacZ 0 huntingtin sh2.4 sh8.2 sh30.1 GFP beta-catenin D GFP sh2.4 sh8.2 sh30.1 Fig. 1. In vitro screening of shRNAs targeting human HD and mouse HDh transcripts. (A) Thirty-five shRNAs (bars above cartoon) targeting conserved sequences (Table S1) spanning human HD and mouse HDh mRNAs were generated with consideration for sequences that promote proper loading of AAVsh8.2-GFP the antisense strands into the RISC. Plasmids expressing U6-driven shRNAs DARPP-32 were transfected into HEK 293 cells, and HD gene silencing was evaluated by QPCR and protein dot blot analyses 48 h after transfection. (B) Three candidate shRNAs targeting sequences in exons 2 (sh2.4), 8 (sh8.2), and 30 (sh30.1) were Iba1 chosen for further study (red bars above cartoon in A). (C) shRNA expression plasmids were transfected into mouse C2C12 cells, and endogenous hunting- Fig. 2. HD shRNAs cause sequence-specific striatal toxicity in mice. (A) tin protein levels were evaluated by Western blot analyses 48 h after trans- Diagram of the recombinant AAV2/1 viral vectors containing shRNA and fection. Mismatch (mis) controls contain 4-bp changes that render the shRNAs hrGFP expression cassettes. (B) Photomicrographs represent the rostral-to- ineffective. ␤-Catenin serves as the loading control. caudal distribution of hrGFP-positive cells in mouse brain after direct injection of virus into the striatum. (Scale bar: 500 ␮m.) (C) QPCR analysis measuring HDh mRNA levels in shRNA-treated mouse striata demonstrates similar silenc- observed in cells throughout the rostral/caudal extent of the stria- ing efficacies among sh2.4, sh8.2, and sh30.1. Mice were injected into the tum and within fibers of the globus pallidus (Fig. 2B). Immuno- striatum with AAVsh2.4-GFP, AAVsh8.2-GFP, AAVsh30.1-GFP, or AAV-GFP, fluorescence analyses indicated that GFP-positive cells colocalized and RNA was harvested 4 months later from GFP-positive striata. All values with a neuronal marker (NeuN), but not with markers for astrocytes were normalized to ␤-actin and are shown relative to AAV-GFP-treated brains. (GFAP) or oligodendrocytes (RIP1) (Fig. S1). QPCR performed (D) Immunohistochemistry reveals that sh2.4 and sh30.1 induce striatal toxicity in mice. Mice were injected with the indicated AAVshRNA-GFP or AAV-GFP on RNA isolated from GFP-positive striatal tissue showed a into the striatum, and histological analyses were performed on brains har- significant and statistically similar reduction of HDh mRNA ex- vested at 4 months after treatment. Representative photomicrographs for pression (Ϸ60%) among the different active shRNA-expressing immunohistochemical staining of DARPP-32-positive neurons (Upper) and vectors, compared with mice injected with AAV-GFP [F(3, 11) ϭ IbaI-positive microglia (Lower) are shown for each treatment group. (Scale 32.3, P Ͻ 0.001 for post hoc analyses comparing each AAV-shRNA bar: 500 ␮m for Upper; 100 ␮m for Lower.) group to the AAV-GFP control] (Fig. 2C). Moreover, Western blot analysis demonstrated a significant reduction in huntingtin protein levels after AAVshRNA-GFP administration, compared with mis- GFP-injected striata demonstrated high Iba1 expression, whereas match controls [t(8) ϭ 3.9, P Ͻ 0.01] (Fig. S2). AAVsh8.2-GFP-injected striata were similar to control mice (Fig. Unexpectedly, immunohistochemical analyses for dopamine- 2D Lower). Moreover, AAVsh2.4-GFP- and AAVsh30.1-GFP- and cAMP-regulated protein (DARPP-32), a marker of medium- injected mice demonstrated dramatic reactive astrogliosis, com- sized spiny projection neurons in the striatum, revealed striatal pared with AAVsh8.2-GFP- and control-injected mice, as evi- toxicity in mice injected with AAVsh2.4-GFP and AAVsh30.1- denced by robust GFAP staining in areas of the striatum

GFP (Fig. 2D Upper). Reduction in DARPP-32 immunoreactivity corresponding to high GFP positivity (data not shown). Notably, a MEDICAL SCIENCES was largely confined to the transduced (GFP-positive) regions of mismatch control for the HD2.4 sequence, AAVsh2.4mis-GFP, the striatum. Interestingly, this toxicity was not seen in mice injected induced toxicity similar to sh2.4 and sh30.1 without reducing HDh with AAVsh8.2-GFP (Fig. 2D Upper). Striata from these mice were mRNA expression. This, in addition to the sh8.2 data, indicates that similar to AAV-GFP-injected control mice. three (two active, one inactive) of four shRNAs were toxic and that To assess whether the observed loss of DARPP-32 staining was toxicity is not caused by silencing huntingtin. associated with microglial activation, tissue sections were stained Although all U6-shRNA expression cassettes were cloned into with an anti-Iba1 antibody to identify both resting and reactive the same viral vector, we tested for the possibility that toxicity microglia throughout the brain. AAVsh2.4-GFP- and AAVsh30.1- correlated with steady-state levels of the expressed products.

McBride et al. PNAS ͉ April 15, 2008 ͉ vol. 105 ͉ no. 15 ͉ 5869 Downloaded by guest on September 27, 2021 inhibitory RNAs, rather than inappropriate sense strand loading A Pos. Control Standard into the RNA-induced silencing complex (RISC) or saturation of shRNA Sense Antisense endogenous RNAi export machinery. sh2.4 Antisense Sequence Levels Are Reduced by Using an Artificial miRNA. Because the toxic shRNAs were expressed at higher levels than the sh8.2 nontoxic, active hairpin, an obvious approach to reduce toxicity would be to lower the viral titer injected. In the brain, decreasing sh30.1 the titers of AAVsh2.4-GFP by a half log (1e12) or a full log (5e11) achieved silencing of HDh mRNA (47% and 51%, respectively), Probe for Antisense Strand but did not alleviate striatal toxicity (Fig. S3). Decreasing the titers even further (1e11 or 5e10) reduced the silencing efficacy to 15% B 150 of controls, an activity level possibly below therapeutic efficacy (Fig. S3). Thus, we tested whether levels of inhibitory RNAs could be minimized without compromising silencing efficacy by using an 100 artificial miRNA as an siRNA shuttle (vs. an shRNA). In corresponding work, we have found that artificial miRNAs 50 effectively silence target gene expression relative to shRNAs with- out generating excessive levels of inhibitory RNAs (R.L.B. and B.L.D., unpublished data). Consequently, we cloned two of the 0 toxic sequences (HD2.4 and HD2.4mis) into an artificial miRNA Relative AS expression Relative sh2.4 sh8.2 sh30.1 AS AS AS scaffold based on human miR-30 (14), thus creating mi2.4 and mi2.4mis (Fig. 4A). We first compared the expression levels of mi2.4 Fig. 3. The nontoxic sh8.2 generates lower levels of processed antisense and sh2.4 by small transcript Northern blot analysis at 48 h after RNA. (A) Small transcript Northern blot was performed to assess AS RNA levels transfection of RNAi-expressing plasmids into HEK 293 cells. present in mouse striata treated with the indicated AAVshRNA-GFP. (Left) Two separately treated striatal tissue samples. (Center and Right) Positive Probing for the HD2.4 antisense strand revealed that mi2.4 pro- controls loaded as standards [10-fold dilutions for both S (Center)orAS(Right) duces substantially lower levels of inhibitory RNAs relative to sh2.4. strands]. (B) Densitometry analysis was used to quantify the relative levels of Notably, sh2.4 generates an abundance of precursor and processed HD AS RNAs. Signals were quantified by using Image J software, and expres- RNAs even at a 10-fold lower dose (Fig. 4B). Despite the dramatic sion is shown as femtomoles per microgram of total RNA. difference in expression levels, mi2.4 reduced endogenous HD transcripts almost as effectively as sh2.4 (50% and 60% silencing, respectively) in HEK 293 cells (Fig. 4C). RNA samples harvested from shRNA-treated striata were an- alyzed by small transcript Northern blot probing for the mature Artificial miRNAs Mitigate Striatal Toxicity in Mice. We next gener- antisense (AS) and sense (S) RNAs generated by the respective ated AAV2/1-expressing mi2.4 or the mi2.4 mismatch control (Figs. shRNAs. Results demonstrate that sh2.4 AS RNA and sh30.1 AS 2B and 4A) to test whether the development of striatal toxicity RNA are expressed more robustly than sh8.2 AS RNA (Fig. 3), could be prevented relative to AAVsh2.4-GFP. Because shRNA- thus correlating toxicity with increased expression levels of the induced toxicity was not dependent on the disease model, subse- shRNAs in vivo. The disparity in expression levels is interesting, quent studies were performed in wild-type mice. Mice were injected particularly given the fact that each shRNA was designed using into the right striatum with AAVsh2.4-GFP, AAVmi2.4-GFP, or the same rules, injected at the same viral dose, driven by the same AAVmi2.4mis-GFP and killed 4 months after injection. The time Pol-III promoter, and silenced HDh mRNA to a similar degree. course, volume, and titer were identical to those used in our earlier Notably, the processed sense strands and unprocessed shRNA shRNA studies (Fig. 2). QPCR performed on RNA isolated from transcripts were not detectable in brain lysates. This finding mouse striata showed a statistically significant reduction of HDh suggests that the toxicity is due, in part, to high levels of mature mRNA (Ϸ70%) after treatment with either sh2.4- or mi2.4-

A B mi2.4 sh2.4 C DNA (ug) 0.222 0.2 1.2

U U 5‘ +1 C C 1.0 GACCGUGUGAAUCAUUGUCUA C sh2.4 CUGGCACACUUAGUAACAGAU U Pre- 3‘-UU A G 0.8 C U 0.6

+1 UAAA 5‘-G...NNN A C G G 0.4 AGUG GCG ACCGUGUGAAUCAUUGUCUAACU C mi2.4 C UCAU CGC UGGCACACUUAGUAACAGAUUGG G A 0.2 3‘-...NNN C A UAGAC 2.4 AS Relative HD mRNA 0 EtBr U6 mi2.4 sh2.4

Fig. 4. An artificial miRNA approach naturally reduces precursor and mature inhibitory RNAs. (A) Sequences and comparison of sh2.4 and mi2.4 containing the core HD2.4 sequence (shaded boxes). Each transcript starts with the ϩ1-G natural to the U6 promoter. The major Drosha and Dicer cleavage sites are shown by hash marks. (B) HEK 293 cells were transfected with HD2.4 RNAi expression plasmids at the indicated amounts, and small-transcript Northern blot was performed 48 h later. Results demonstrate that sh2.4 generates abundant levels of unprocessed precursor (Pre-) and processed antisense RNAs (2.4AS) even at a 10-fold-lower dose relative to mi2.4. Ethidium bromide (EtdBr) staining is shown as the loading control. (C) HD2.4 RNAi expression plasmids were transfected into HEK 293 cells, and QPCR analysis was performed 48 h later to measure endogenous HD mRNA levels. Results demonstrate that mi2.4 silences HD transcripts efficiently, relative to sh2.4, despite being expressed at considerably lower levels.

5870 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0801775105 McBride et al. Downloaded by guest on September 27, 2021 A B HD2.4 antisense strand, demonstrated considerably more mature 1.2 5 HD2.4 antisense RNAs in sh2.4-treated mice relative to mi2.4- 1.0 4 treated mice (Fig. 5C). These results corroborate our in vitro 0.8 findings and correlate the improvement in toxicity with reduced 3 0.6 NS levels of HD2.4 antisense RNA. 2 We further assessed striatal toxicity by histological analyses. 0.4 Immunolabeling for DARPP-32 expression revealed significant 1 0.2 attenuation of striatal toxicity in AAVmi2.4-GFP-injected cohorts Relative HDh mRNA 0 Relative CD11b mRNA 0 relative to AAVsh2.4-GFP-injected mice (Fig. 5D Middle). More- mi2.4 mi2.4 sh2.4 mi2.4 mi2.4 sh2.4 mis mis over, the intense microglial activation (Iba1-positive cells) seen in AAVsh2.4-GFP-injected mice was scarcely present in AAVmi2.4- C mi2.4 GFP-injected mice (Fig. 5D Bottom and Fig. S4). Of note, mi2.4mis- mis mi2.4 sh2.4 treated brains also showed no apparent toxicity by these analyses, 2.4 AS whereas HD2.4mis was toxic when delivered as an shRNA (data not shown). Thus, sequences encoding HD2.4 and HD2.4mis were toxic EtBr in the setting of an shRNA in the brain, but not in the context of a miRNA scaffold. D mi2.4 mis mi2.4 sh2.4 Discussion Here, we show that some shRNAs cause toxicity in mouse striatum independent of HDh mRNA silencing. Similar to our work, Grimm and colleagues (15) observed acute liver toxicity and mortality in hrGFP mice after systemic shRNA delivery, which correlated with in- creased mature antisense RNA levels. However, there are impor- tant differences between our findings. First, Grimm et al. found that lowering the vector dose by Ϸ10-fold significantly improved the lethal effects of some shRNAs on liver function and animal

DARPP-32 viability. In our studies, reducing the dose led to lower transduction throughout the striatum, but did not abrogate toxicity. Second, the data by Grimm and colleagues show significant buildup of shRNA precursors in liver cells. They attributed the liver toxicity, in part, to

Iba1 the saturation of endogenous RNAi export machinery. In our work, we detected abundant levels of unprocessed shRNAs in vitro, but, interestingly, low to undetectable levels in vivo. This finding suggests Fig. 5. Artificial miRNAs mitigate striatal toxicity in mice. (A and B) QPCR that export was likely not limiting in our studies. Alternatively, the analyses were performed to measure mouse HDh (A) and CD11b (B) mRNA striatal toxicity may be caused by the buildup of antisense RNAs levels in AAV-RNAi-injected striata harvested 4 months after treatment (NS, and subsequent off-target silencing of unintended mRNAs. Our not significant). Samples were normalized to ␤-actin. Results, shown relative data on sh8.2 also are consistent with this; sh8.2 was not toxic when to uninjected striata, demonstrate that mi2.4 silences HD transcripts as effec- delivered at the same dose as sh2.4 and sh30.1. Although silencing tively as sh2.4, but avoids induction of CD11b, a marker for microglial activa- tion. (C) Small-transcript Northern blot analysis for mature HD2.4 AS RNAs activity was similar among the three shRNAs, levels of mature present in AAV-RNAi-treated striatal lysates reveals a robust disparity be- product for sh8.2 were significantly lower. tween the levels generated from sh2.4 and mi2.4 vectors. EtdBr staining is We found that moving the HD2.4 and HD2.4mis sequences, both shown as the loading control. (D) Histological analyses demonstrate the of which caused toxicity in the context of a shRNA, into a miRNA improved safety profile of mi2.4. Mice were injected with the indicated scaffold significantly reduced neurotoxicity within the striatum with AAV-RNAi-GFP viruses into the striatum, and histological analyses were per- no sacrifice in gene-silencing efficacy. We correlated this positive formed on brains harvested at 4 months after treatment. Photomicrographs effect to lower steady-state levels of mature antisense RNAs representing hrGFP (Top), immunohistochemical staining of DARPP-32- processed from the artificial mi2.4 relative to sh2.4. Whether this positive neurons (Middle), and IbaI-positive microglia (Bottom) are shown for disparity in expression levels results from the differences in tran- each treatment group. (Scale bar: 500 ␮m.) scription or the stability between shRNAs and artificial miRNAs remains unknown. However, the latter provides a more likely expressing vectors, compared with uninjected striata or striata explanation because sh2.4 and mi2.4 are expressed from the same Ϸ treated with mi2.4mis [F(2, 8) ϭ 77.6, P Ͻ 0.001 for post hoc mouse U6 promoter and only differ in size by 100 . analyses comparing sh2.4 and mi2.4 vs. uninjected and mi2.4mis] In addition to improved safety profiles, artificial miRNAs are (Fig. 5A). Importantly, the degree of HDh mRNA silencing be- amenable to Pol-II-mediated transcription. Conversely, shRNAs have limited spacing flexibility for expressing shRNAs from tween sh2.4 and mi2.4 was similar and not significantly different Pol-II-based promoters (16). This advantage of miRNA-based (P Ͼ 0.05). Additional QPCR analyses were performed on these systems allows for regulated and cell-specific expression of samples to measure CD11b mRNA, a readout for microglial inhibitory RNAs. These versatile expression strategies advance activation, as an initial assessment for toxicity. Striata treated with the application of artificial miRNAs as biological tools and may sh2.4 showed nearly a 4-fold increase of CD11b mRNA relative to further limit potential toxicity in therapeutic applications. MEDICAL SCIENCES uninjected striata, whereas mi2.4- and mi2.4mis-treated striata In some diseases, it is possible to specifically target disease- ϭ Ͻ showed only minimal induction [F(2, 8) 23.6, P 0.001 for post linked SNPs that exist on the mutant transcript (17, 18). For HD, hoc analyses comparing sh2.4 to all other groups] (Fig. 5B). To however, no prevalent SNP has been reported. Because earlier determine whether these differences in toxicity could be attributed work showed that a minimum of 50% huntingtin expression is to levels of HD2.4 inhibitory RNAs, we performed Northern blot required to offset the embryonic lethality noted in huntingtin- analysis on the same RNA samples used for the QPCR analyses. null mice (19), knowing the consequences of reducing huntingtin Although silencing efficacies between the sh2.4- and mi2.4-treated expression in adult brain is important to moving non-allele- groups were comparable, Northern blot analysis, probing for the specific RNAi forward as a HD therapy. Our data with sh8.2 and

McBride et al. PNAS ͉ April 15, 2008 ͉ vol. 105 ͉ no. 15 ͉ 5871 Downloaded by guest on September 27, 2021 mi2.4 are encouraging and suggest that the mammalian brain can for all studies were 0.2 ␮l/min. Mice used in histological analyses were anes- tolerate Ͼ50% reduction in HD mRNA for 4 months, the last thetized with a ketamine/xylazine mix and transcardially perfused with 20 ml time point studied. The long-term safety and efficacy of sh8.2 is of 0.9% cold saline, followed by 20 ml of 4% paraformaldehyde in 0.1 M PO4 ␮ currently being tested in a study including histochemical, bio- buffer. Brains were removed and postfixed overnight, and 40- m thick sec- tions were collected. Mice used for molecular analyses were perfused with 20 chemical, and behavioral readouts in CAG140 HD mice. ml of 0.9% cold saline, and brain was removed and blocked into 1-mm-thick In summary, we show that reducing HDh mRNA levels in adult coronal slices. Tissue punches were taken by using a tissue corer (1.4 mm in mammalian brain is tolerated. We also make the important obser- diameter). All tissue punches were flash frozen in liquid nitrogen and stored vation that the toxicity of shRNAs after their expression in brain at Ϫ80°C until used. could be alleviated by moving the inhibitory RNA sequences into an artificial miRNA scaffold. Thus, miRNA-based approaches are Molecular Studies. For in vitro shRNA screening, shRNA expression plasmids were more suitable for achieving RNAi in the brain to address basic transfected (Lipofectamine 2000; Invitrogen) into human HEK 293 cells or mouse research questions or develop disease therapies. C2C12 cells, which naturally express full-length human or mouse huntingtin, respectively. Huntingtin levels were assessed by protein dot blot (anti-huntingtin Materials and Methods primary antibody MAB2166, 1:5,000; Chemicon) or Western blot (protein loading control, anti-␤-catenin, 1:4,000; AbCam). Knockdown also was assessed by QPCR Expression Vectors and AAV. shRNA expression cassettes were generated by PCR using a human huntingtin-specific TaqMan primer/probe set with normalization as described (8) and cloned into pCR-Blunt-II TOPO vectors (Invitrogen). Each to a human GAPDH primer/probe set. This QPCR strategy also was used to candidate shRNA expression cassette consisted of a mouse U6 promoter, an evaluate HD knockdown mediated by sh2.4 and mi2.4 in Fig. 4B. shRNA [targeting huntingtin sequences, mismatch control sequences (containing For in vivo QPCR analyses, tissue was dissected from GFP-positive striatum, and four changes relative to the respective huntingtin shRNAs), or E. coli relative gene expression was assessed by using TaqMan primer/probe sets for ␤-gal; shLacZ], and an RNA polymerase III termination sequence (six thymidine mouse HDh, CD11b, and ␤-actin. All values were quantified by using the ⌬⌬CT nucleotides). For artificial miRNAs, siRNA sequences based on HD2.4 or HD2.4mis method (normalizing to ␤-actin) and calibrated to either AAV-GFP-injected stri- were embedded into an artificial miRNA scaffold comparable to human miR-30 ata (screening study) or uninjected striata (miRNA-shRNA comparison study). to generate mi2.4 and mi2.4mis (general structure shown in Fig. 4A). The artificial For Northern blot analyses, tissue was dissected from GFP-positive striatum. Ͼ miRNA stem loops were cloned into a mouse U6 expression vector so that 30 nt RNA was harvested by TRIzol reagent and RNA (1–5 ␮g and 15 ␮g for in vivo and Ј Ј (5 and 3 ) flank the stem loop in the transcribed product. in vitro studies, respectively) was resolved on 15% polyacrylamide/urea gels, and AAV shuttle plasmids pAAVsh2.4-GFP, pAAVsh2.4mis-GFP, pAAVsh8.2- RNA was visualized by ethidium bromide staining and UV exposure to assess GFP, pAAVsh30.1-GFP, pAAVmi2.4-GFP, and pAAVmi2.4mis-GFP contain the loading and RNA quality. Samples were then transferred to Hybond-Nϩ/XL respective RNAi expression cassettes driven by the mouse U6 promoter. The membranes (Amersham Pharmacia) and UV cross-linked. Blots were probed with AAV shuttles also contained a hrGFP gene under the control of the human 32P-labeled at 30–36°C overnight, washed in 2ϫ SSC at 30–36°C, cytomegalovirus immediate-early gene enhancer/promoter region, a chimeric and exposed to film. ␤ human -globin eGFP expression cassette followed by the splice donor/human For in vivo Western blot analysis, tissue was dissected from GFP-positive Ig splice acceptor site, and a bovine growth hormone poly (A) signal. These striatum and lysed in 150 ␮l of lysis buffer, and protein level was quantified with transcriptional units are flanked at each end by AAV serotype 2 145-bp the DC protein assay (Bio-Rad). Then 10 ␮g of total protein was separated on 8% inverted terminal repeat sequences. The transpackaging plasmids, SDS polyacrylamide gel before transferring to a 0.45-␮m PVDF membrane. The pBSHSPR2C1, were constructed as follows: genomic DNA was extracted from membrane was blocked with 2% milk in PBS-Tween 20 (0.05%) and incubated AAV1 (American Type Culture Collection), and the cap coding sequence was with either an anti-huntingtin antibody (1:5,000; Chemicon) or an anti-␤-actin amplified by PCR using Pfx polymerase (Invitrogen). The AAV2 cap gene was antibody (1:10,000; Sigma), followed by a conjugated goat anti-mouse secondary excised from the AAV2 helper pBSHSPRC2.3 and replaced with the antibody (1:10,000; Jackson ImmunoResearch) and an ECL-Plus substrate (Amer- amplified AAV1 cap sequence by using a Swa I restriction site in the rep/cap sham Biosciences), and then exposed to film. intergenic junction and a BsrG I site engineered just upstream of the AAV2 poly(A) signal. The resulting transpackaging construct, pBSHSPR2C1, contains Immunohistochemical Analyses. Briefly, 40-␮m-thick, free-floating coronal brain the AAV2 rep gene under the control of a minimal eukaryotic promoter and sections were processed for immunohistochemical visualization of striatal neu- the AAV1 cap ORF positioned between the AAV2 rep/cap intergenic junction rons (DARPP-32, 1:100; Cell Signaling Technology) and microglia (Iba1, 1:1,000; and the AAV2 poly(A) signal. The plasmid pAd Helper 4.1 expresses the E2a, WAKO) by using the biotin-labeled antibody procedure. Primary antibody incu- E4-orf6, and VA genes of adenovirus type 5 (Ad5) for AAV amplification. bations were carried out for 24 h at room temperature. Sections were incubated Recombinant AAV vectors were produced by a standard calcium phosphate in goat anti-rabbit biotinylated IgG secondary antibodies (1:200; Vector Labora- transfection method in HEK 293 cells by using the Ad helper, transpackaging, and tories) for1hatroom temperature. In all staining procedures, deletion of the AAV shuttle plasmids as described (20). Vector titers were determined by real- primary antibody served as a control. Sections were mounted onto Superfrost ϫ 12 time PCR and were between 5 and 20 10 DNase-resistant particles per ml. Plus slides and coverslipped with Gelmount (Biomeda). Images were captured by Vector infectivity was assessed in a TCID50 assay by using the HeLa-based B50 cell using an Olympus BX60 light microscope and DP70 digital camera, along with an line (21). Olympus DP Controller software.

Animals. All animal protocols were approved by the Institutional Animal Care and Statistical Analyses. All statistical analyses were performed by using SigmaStat Use Committee at the University of Iowa. CAG140 heterozygous knockin mice statistical software (SYSTAT). QPCR analyses for huntingtin and CD11b expression (10) and wild-type littermates were bred and maintained in the animal vivarium were performed by using a one-way ANOVA, as was Northern blot densitometry at the University of Iowa. Mice were genotyped and repeat length identified by analysis. Upon a significant effect, Bonferroni post hoc analyses were performed separate PCRs using primers flanking the CAG repeat. Mice were housed in to assess for significant differences between individual groups. Western blot groups of either two or three per cage and in a controlled temperature environ- densitometry analysis was performed by using a two-tailed Student’s t test. In all ment on a 12-h light/dark cycle. Food and water were provided ad libitum. cases, P Ͻ 0.05 was considered significant.

AAV Injections. CAG140 knockin or wild-type mice were injected with AAVsh- Figure Preparation. All photographs were formatted with Adobe Photoshop RNAs or AAV-miRNAs (at the indicated titer) at 5 weeks of age and killed at 4 software, all graphs were made with Prism Graph software, and all figures were months after injection. Procedures were performed as reported previously (8) constructed with Adobe Illustrator software. with the following exceptions. In the initial study, 5-␮l injections of either AAVsh2.4GFP, AAV30.1sh-GFP, AAVsh8.2-GFP, or AAV-GFP were made bilat- ACKNOWLEDGMENTS. We thank the B.L.D. and McCray laboratories for feed- Ϯ erally into striata (coordinates: 0.86 mm rostral to bregma, 1.8 mm lateral to back and discussion. This work was supported by National Institutes of Health midline, 3.5 mm ventral to the skull surface). For the miRNA/shRNA compar- Grants NS-50210, HD-44093, DK-54759, and NS-592372; the Hereditary Disease ison study, 5-␮l injections of vector were injected unilaterally. Injection rates Foundation; and the Roy J. Carver Trust.

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