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DNA-directed RNA interference
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<h2 id="ddrnai-mechanism">ddRNAi mechanism</h2> <p>Unlike <a href="/facts/Small_interfering_RNA/891ytkD9">small interfering RNAs</a> (siRNA) that turn over within a cell and silence genes transiently, DNA constructs are continually transcribed, replenishing the cellular supply of siRNAs. This allows for the long-term silencing of targeted genes, which has the potential for ongoing clinical benefit with reduced medical intervention.<a class="footnote-ref" id="fnref:9" href="#fn:9"><sup>9</sup></a><a class="footnote-ref" id="fnref:10" href="#fn:10"><sup>10</sup></a> </p> <h2 id="organization-of-ddrnai-constructs">Organization of ddRNAi constructs</h2> <p>Figure 1 illustrates the most common type of ddRNAi DNA construct designed to express a shRNA. This figure consists of a <a href="/facts/Promoter_(genetics)/YDYBzLfg">promoter</a> sequence driving the expression of <a href="/facts/Sense_(molecular_biology)/hcKgNvy6">sense</a> and <a href="/facts/Antisense/hcKgNvy6">antisense</a> sequences separated by a loop sequence, followed by a transcriptional <a href="/facts/Terminator_(genetics)/mdR46gBa">terminator</a>. The antisense sequence processed from the shRNA can bind to the target RNA and specify its degradation. shRNA constructs typically encode sense and antisense sequences of 20–30 <a href="/facts/Nucleotide/pQKEwlJI">nucleotides</a>. Flexibility in construct design is possible; for example, the positions of sense and antisense sequences can be reversed, and other modifications and additions can alter intracellular shRNA processing.<a class="footnote-ref" id="fnref:11" href="#fn:11"><sup>11</sup></a> Moreover, a variety of promoter loop and terminator sequences can be used.<a class="footnote-ref" id="fnref:12" href="#fn:12"><sup>12</sup></a> </p> <p>A variant of this is the <a href="/facts/Gene_cassette/VeqfwJ62">multi-cassette</a> (Figure 2b). Designed to express two or more shRNAs, it can target multiple sequences for degradation simultaneously, which can be beneficial in circumstances such as when targeting viruses. Natural sequence variations can render a single shRNA-target site unrecognizable, preventing RNA degradation. Multi-cassette constructs that target multiple sites within the same viral RNA circumvent this issue.<a class="footnote-ref" id="fnref:13" href="#fn:13"><sup>13</sup></a> </p> <h2 id="delivery">Delivery</h2> <p>Delivery of ddRNAi DNA constructs is a major challenge for RNAi-based therapy. There are a number of clinically-approved <a href="/facts/Gene_therapy/CaxRLfKM">gene therapy</a> vectors developed for therapeutic use. Two broad strategies to facilitate the delivery of DNA constructs to the desired cells are available: these use either viral vectors or one of several classes of <a href="/facts/Transfection/R94fLSQF">transfection</a> reagents. </p><p><i><a href="/facts/In_vivo/kWcWyl6f">In vivo</a> </i>delivery of ddRNAi constructs has been demonstrated using a range of vectors and reagents with different <a href="/facts/Route_of_administration/LsyoHTzH">routes of administration</a> (ROA).<a class="footnote-ref" id="fnref:14" href="#fn:14"><sup>14</sup></a><a class="footnote-ref" id="fnref:15" href="#fn:15"><sup>15</sup></a><a class="footnote-ref" id="fnref:16" href="#fn:16"><sup>16</sup></a><a class="footnote-ref" id="fnref:17" href="#fn:17"><sup>17</sup></a> ddRNAi constructs have also been successfully delivered into host cells <a href="/facts/Ex_vivo/bs3kVn5h">ex vivo</a>, and then transplanted back into the host.<a class="footnote-ref" id="fnref:18" href="#fn:18"><sup>18</sup></a><a class="footnote-ref" id="fnref:19" href="#fn:19"><sup>19</sup></a><a class="footnote-ref" id="fnref:20" href="#fn:20"><sup>20</sup></a> </p><p>For example, in a phase I clinical trial at the <a href="/facts/City_of_Hope_National_Medical_Center/jCR06v7r">City of Hope National Medical Center</a>, California, four HIV-positive patients with <a href="/facts/Non-Hodgkin_lymphoma/KIFxt74F">non-Hodgkin's lymphoma</a> were successfully treated with autologous <a href="/facts/Hematopoietic_stem_cell/RQNE9IRu">hematopoietic progenitor cells</a> pre-transduced ex vivo with ddRNAi constructs using <a href="/facts/Lentiviral_vector_in_gene_therapy/mSCu2uLu">lentiviral vectors</a>. This construct was designed to express three therapeutic RNAs, one of which was a shRNA, thereby combating HIV replication in three different ways:<a class="footnote-ref" id="fnref:21" href="#fn:21"><sup>21</sup></a> </p> <ul><li>shRNA, silencing the tat and rev genes of the HIV genome;</li> <li>CCR5 ribozyme, inhibiting viral cell entry;</li> <li>TAR decoy RNA, inhibiting initiation of viral transcription.</li></ul> <p>Ongoing expression of the shRNA has been confirmed in <a href="/facts/T_cell/5z6PQ9UN">T cells</a>, <a href="/facts/Monocyte/FW4NNMKM">monocytes</a>, and <a href="/facts/B_cell/6Y1tgwwp">B cells</a> more than one year after transplantation.<a class="footnote-ref" id="fnref:22" href="#fn:22"><sup>22</sup></a> </p> <h2 id="therapeutic-applications">Therapeutic applications</h2> <h3>Neuropathic pain</h3> <p>Nervana is an investigational V construct that <a href="/facts/Gene_knockdown/jgJjphvm">knocks down</a> the expression of <a href="/facts/Protein_kinase_C/6WSQgAWv">protein kinase C</a> gamma (PKCγ) known to be associated with <a href="/facts/Neuropathic_pain/PAGm7Vhd">neuropathic pain</a> and <a href="/facts/Morphine/8ngpXHDc">morphine tolerance</a>.<a class="footnote-ref" id="fnref:23" href="#fn:23"><sup>23</sup></a> </p><p>Two conserved PKCγ sequences across all key model species and humans have been identified, and both single and double DNA cassettes are designed. <a href="/facts/In_vitro/evp3hAX6">In vitro</a>, the expression of PKCγ was silenced by 80%. When similar ddRNAi constructs were delivered intrathecally using a lentiviral vector, pain relief in a neuropathic-rat model was demonstrated.<a class="footnote-ref" id="fnref:24" href="#fn:24"><sup>24</sup></a> </p> <h3>Drug-resistant non-small-cell lung cancer</h3> <p>The development of resistance to <a href="/facts/Chemotherapy/fWTLbaUT">chemotherapies</a> such as <a href="/facts/Paclitaxel/sGxTE647">paclitaxel</a> and <a href="/facts/Cisplatin/eXLc7Fzw">cisplatin</a> in <a href="/facts/Non-small-cell_lung_cancer/9T47knRp">non-small-cell lung cancer</a> (NSCLC) is strongly associated with overexpression of <a href="/facts/Class_III_b-tubulin/8qoz8sGh">beta III tubulin</a>. Investigations by the Children's Cancer Institute Australia (<a href="/facts/UNSW_Lowy_Cancer_Research_Centre/nEvkJJrr">University of New South Wales, Lowy Cancer Research Centre</a>) demonstrated that beta III-tubulin knockdown by ddRNAi delayed <a href="/facts/Tumor/K0ytzmPz">tumor</a> growth and increased <a href="/facts/Chemosensitivity_assay/pBVs9pwX">chemosensitivity</a> in mouse models.<a class="footnote-ref" id="fnref:25" href="#fn:25"><sup>25</sup></a> </p><p>Tributarna is a triple DNA cassette expressing three shRNA molecules each of which separately targets beta III tubulin and strongly inhibits its expression. Studies in an orthotopic-mouse model, where the construct is delivered by a modified <a href="/facts/Polyethylenimine/BXhSol9h">polyethylenimine</a> vector, jetPEI, that targets lung tissue are in progress. </p> <h3>Hepatitis B viral infection</h3> <p>The <a href="/facts/Hepatitis_B_virus/vk2qncf2">hepatitis B virus</a> (HBV) genome encodes its own <a href="/facts/DNA_polymerase/xtNLncBu">DNA polymerase</a> for replication. Biomics Biotechnologies has evaluated around 5000 siRNA sequences of this gene for effective knockdown; five sequences were chosen for further investigation and were shown to have silencing activity when converted into shRNA expression cassettes. A multi-cassette construct, Hepbarna, is under preclinical development for delivery by an <a href="/facts/Adeno-associated_virus/zUxAB45G">adeno-associated virus</a> 8 (AAV-8) liver-targeting vector. </p> <h3>Oculopharyngeal muscular dystrophy</h3> <p>Classified as an <a href="/facts/Orphan_disease/Nu1G7rEw">orphan disease</a>, there is currently no therapy for <a href="/facts/Oculopharyngeal_muscular_dystrophy/4Tm3xvL8">oculopharyngeal muscular dystrophy</a> (OPMD), as it is caused by a mutation in the <a href="/facts/PABPN1/rDg2oEce">poly(A) binding protein nuclear 1</a> (PABPN1) gene. Silencing the mutant gene using ddRNAi offers a potential therapeutic approach.<a class="footnote-ref" id="fnref:26" href="#fn:26"><sup>26</sup></a> </p> <h3>HIV/AIDS</h3> <p>Besides the ex vivo approach discussed above, the Center for Infection and Immunity Amsterdam (CINIMA) of the <a href="/facts/University_of_Amsterdam/VqQ6q6QK">University of Amsterdam</a>, Netherlands, is extensively researching the therapeutic potential of multi-cassette DNA constructs for HIV.<a class="footnote-ref" id="fnref:27" href="#fn:27"><sup>27</sup></a> </p> <h2 id="concerns">Concerns</h2> <p>As with all <a href="/facts/Gene_therapy/CaxRLfKM">gene therapies</a>, a number of safety and toxicity issues must be evaluated during the development of ddRNAi therapeutic techniques. </p> <h3>Oncogene activation by viral insertion</h3> <p>Some gene therapy vectors integrate into the host genome, thereby acting as insertional <a href="/facts/Mutagen/xI5lKvmp">mutagens</a>. This was a particular issue with early <a href="/facts/Retrovirus/GWAx3C3o">retroviral vectors</a>, where insertions adjacent to <a href="/facts/Oncogenes/WLFu6Ma5">oncogenes</a> resulted in the development of <a href="/facts/Lymphatic_system/yw5QLgMT">lymphoid</a> tumors.<a class="footnote-ref" id="fnref:28" href="#fn:28"><sup>28</sup></a> </p><p>Adeno-associated virus (AAV) vectors are considered low-risk for host-genome integration, as AAV infection has not been associated with the induction of cancers in humans despite widespread prevalence across the general population. Moreover, extensive clinical use of AAV vectors has provided no evidence of <a href="/facts/Carcinogenicity/soH8Y9CE">carcinogenicity</a>. While lentiviral vectors do integrate into the genome, they do not appear to show a propensity to activate oncogene expression.<a class="footnote-ref" id="fnref:29" href="#fn:29"><sup>29</sup></a> </p> <h3>Immune response to gene therapy vectors</h3> <p>An <a href="/facts/Immune_response/dtLulqIy">immunological response</a> to an adenoviral vector resulted in the death of a patient in an early human trial. Careful monitoring of potential toxicities in preclinical testing and analysis of any pre-existing antibodies to gene therapy vectors in patients minimizes such risks. </p> <h3>Innate immune response</h3> <p>siRNAs have been shown to activate immune responses through interaction with <a href="/facts/Toll-like_receptor/hj4qIWP8">toll-like receptors</a> (TLRs), leading to <a href="/facts/Interferon/63XfwpEh">interferon</a> responses. These TLRs reside on the cell's outer surface, so ddRNAi constructs, which are delivered directly into the intracellular space, are not expected to induce such a response.<a class="footnote-ref" id="fnref:30" href="#fn:30"><sup>30</sup></a> </p> <h3>Toxic effects due to over-expression of shRNAs</h3> <p>High-level expression of shRNAs has been shown to be toxic. Strategies to minimize levels of shRNA expression<a class="footnote-ref" id="fnref:31" href="#fn:31"><sup>31</sup></a> or promote precise processing of shRNAs<a class="footnote-ref" id="fnref:32" href="#fn:32"><sup>32</sup></a> can mitigate this issue. </p> <h3>Off-target effects</h3> <p>The unintended silencing of genes that share <a href="/facts/Sequence_homology/mCIVStrA">sequence homology</a> with expressed shRNAs could theoretically occur.<a class="footnote-ref" id="fnref:33" href="#fn:33"><sup>33</sup></a> Careful selection of shRNA sequences and thorough preclinical testing of constructs can mitigate this risk. </p> <h2 id="further-reading">Further reading</h2> <ul><li>Rice, R. R.; Muirhead, A. N.; Harrison, B. T.; Kassianos, A. J.; Sedlak, P. L.; Maugeri, N. J.; Goss, P. J.; Davey, J. R.; James, D. E.; Graham, M. W. (2005). "Simple, Robust Strategies for Generating DNA-Directed RNA Interference Constructs". <i>RNA Interference</i>. Methods in Enzymology. Vol. 392. pp. 405–419. <a href="/facts/Doi_(identifier)/muM9Etpq">doi</a>:<a href="https://doi.org/10.1016%2FS0076-6879%2804%2992024-1">10.1016/S0076-6879(04)92024-1</a>. <a href="/facts/ISBN_(identifier)/15AdSPa9">ISBN</a> 9780121827977. <a href="/facts/PMID_(identifier)/JlHAvMHt">PMID</a> <a href="https://pubmed.ncbi.nlm.nih.gov/15644195">15644195</a>.</li> <li>Liu, Y. P.; Westerink, J. T.; Brake, O.; Berkhout, B. (2011). "RNAi-Inducing Lentiviral Vectors for Anti-HIV-1 Gene Therapy". <i>Antiviral RNAi</i>. Methods in Molecular Biology. Vol. 721. pp. 293–311. <a href="/facts/Doi_(identifier)/muM9Etpq">doi</a>:<a href="https://doi.org/10.1007%2F978-1-61779-037-9_18">10.1007/978-1-61779-037-9_18</a>. <a href="/facts/ISBN_(identifier)/15AdSPa9">ISBN</a> 978-1-61779-036-2. <a href="/facts/PMID_(identifier)/JlHAvMHt">PMID</a> <a href="https://pubmed.ncbi.nlm.nih.gov/21431693">21431693</a>.</li></ul> <h2 id="references">References</h2> <ol> <li id="fn:1"><p>Lambeth, L. S.; Smith, C. A. (2013). "Short Hairpin RNA-Mediated Gene Silencing". SiRNA Design. Methods in Molecular Biology. Vol. 942. pp. 205–232. doi:10.1007/978-1-62703-119-6_12. ISBN 978-1-62703-118-9. PMID 23027054. <a href="978-1-62703-118-9" target="_blank">978-1-62703-118-9</a> <a href="#fnref:1" class="footnote-back-ref">↩</a></p></li> <li id="fn:2"><p>Digiusto, D. L.; Krishnan, A.; Li, L.; Li, H.; Li, S.; Rao, A.; Mi, S.; Yam, P.; Stinson, S.; Kalos, M.; Alvarnas, J.; Lacey, S. F.; Yee, J. -K.; Li, M.; Couture, L.; Hsu, D.; Forman, S. J.; Rossi, J. J.; Zaia, J. A. (2010). "RNA-Based Gene Therapy for HIV with Lentiviral Vector-Modified CD34+ Cells in Patients Undergoing Transplantation for AIDS-Related Lymphoma". Science Translational Medicine. 2 (36): 36ra43. doi:10.1126/scitranslmed.3000931. PMC 3130552. PMID 20555022. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3130552" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3130552</a> <a href="#fnref:2" class="footnote-back-ref">↩</a></p></li> <li id="fn:3"><p>Chen, C. -C.; Chang, C. -M.; Sun, C. -P.; Yu, C. -P.; Wu, P. -Y.; Jeng, K. -S.; Hu, C. -P.; Chen, P. -J.; Wu, J. -C.; Shih, C. H.; Gershwin, M. E.; Tao, M. -H. (2011). "Use of RNA interference to modulate liver adenoma development in a murine model transgenic for hepatitis B virus". Gene Therapy. 19 (1): 25–33. doi:10.1038/gt.2011.60. PMID 21562593. <a href="https://doi.org/10.1038%2Fgt.2011.60" target="_blank">https://doi.org/10.1038%2Fgt.2011.60</a> <a href="#fnref:3" class="footnote-back-ref">↩</a></p></li> <li id="fn:4"><p>Suhy, D. A.; Kao, S. C.; Mao, T.; Whiteley, L.; Denise, H.; Souberbielle, B.; Burdick, A. D.; Hayes, K.; Wright, J. F.; Lavender, H.; Roelvink, P.; Kolykhalov, A.; Brady, K.; Moschos, S. A.; Hauck, B.; Zelenaia, O.; Zhou, S.; Scribner, C.; High, K. A.; Renison, S. H.; Corbau, R. (2012). "Safe, Long-term Hepatic Expression of Anti-HCV shRNA in a Nonhuman Primate Model". Molecular Therapy. 20 (9): 1737–1749. doi:10.1038/mt.2012.119. PMC 3437581. PMID 22735378. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3437581" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3437581</a> <a href="#fnref:4" class="footnote-back-ref">↩</a></p></li> <li id="fn:5"><p>McCarroll, J. A.; Gan, P. P.; Liu, M.; Kavallaris, M. (2010). "III-Tubulin is a Multifunctional Protein Involved in Drug Sensitivity and Tumorigenesis in Non-Small Cell Lung Cancer". Cancer Research. 70 (12): 4995–5003. doi:10.1158/0008-5472.CAN-09-4487. PMID 20501838. <a href="/wiki/Maria_Kavallaris" target="_blank">/wiki/Maria_Kavallaris</a> <a href="#fnref:5" class="footnote-back-ref">↩</a></p></li> <li id="fn:6"><p>Zou, W.; Song, Z.; Guo, Q.; Liu, C.; Zhang, Z.; Zhang, Y. (2011). "Intrathecal Lentiviral-Mediated RNA Interference Targeting PKCγ Attenuates Chronic Constriction Injury–Induced Neuropathic Pain in Rats". Human Gene Therapy. 22 (4): 465–475. doi:10.1089/hum.2010.207. PMID 21087146. <a href="/wiki/Doi_(identifier)" target="_blank">/wiki/Doi_(identifier)</a> <a href="#fnref:6" class="footnote-back-ref">↩</a></p></li> <li id="fn:7"><p>Senzer, N.; Barve, M.; Kuhn, J.; Melnyk, A.; Beitsch, P.; Lazar, M.; Lifshitz, S.; Magee, M.; Oh, J.; Mill, S. W.; Bedell, C.; Higgs, C.; Kumar, P.; Yu, Y.; Norvell, F.; Phalon, C.; Taquet, N.; Rao, D. D.; Wang, Z.; Jay, C. M.; Pappen, B. O.; Wallraven, G.; Brunicardi, F. C.; Shanahan, D. M.; Maples, P. B.; Nemunaitis, J. (2011). "Phase I Trial of "bi-shRNAifurin/GMCSF DNA/Autologous Tumor Cell" Vaccine (FANG) in Advanced Cancer". Molecular Therapy. 20 (3): 679–686. doi:10.1038/mt.2011.269. PMC 3293620. PMID 22186789. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3293620" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3293620</a> <a href="#fnref:7" class="footnote-back-ref">↩</a></p></li> <li id="fn:8"><p>Millington-Ward, S.; Chadderton, N.; O'Reilly, M.; Palfi, A.; Goldmann, T.; Kilty, C.; Humphries, M.; Wolfrum, U.; Bennett, J.; Humphries, P.; Kenna, P. F.; Farrar, G. J. (2011). "Suppression and Replacement Gene Therapy for Autosomal Dominant Disease in a Murine Model of Dominant Retinitis Pigmentosa". Molecular Therapy. 19 (4): 642–649. doi:10.1038/mt.2010.293. PMC 3070095. PMID 21224835. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3070095" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3070095</a> <a href="#fnref:8" class="footnote-back-ref">↩</a></p></li> <li id="fn:9"><p>Digiusto, D. L.; Krishnan, A.; Li, L.; Li, H.; Li, S.; Rao, A.; Mi, S.; Yam, P.; Stinson, S.; Kalos, M.; Alvarnas, J.; Lacey, S. F.; Yee, J. -K.; Li, M.; Couture, L.; Hsu, D.; Forman, S. J.; Rossi, J. J.; Zaia, J. A. (2010). "RNA-Based Gene Therapy for HIV with Lentiviral Vector-Modified CD34+ Cells in Patients Undergoing Transplantation for AIDS-Related Lymphoma". Science Translational Medicine. 2 (36): 36ra43. doi:10.1126/scitranslmed.3000931. PMC 3130552. PMID 20555022. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3130552" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3130552</a> <a href="#fnref:9" class="footnote-back-ref">↩</a></p></li> <li id="fn:10"><p>Suhy, D. A.; Kao, S. C.; Mao, T.; Whiteley, L.; Denise, H.; Souberbielle, B.; Burdick, A. D.; Hayes, K.; Wright, J. F.; Lavender, H.; Roelvink, P.; Kolykhalov, A.; Brady, K.; Moschos, S. A.; Hauck, B.; Zelenaia, O.; Zhou, S.; Scribner, C.; High, K. A.; Renison, S. H.; Corbau, R. (2012). "Safe, Long-term Hepatic Expression of Anti-HCV shRNA in a Nonhuman Primate Model". Molecular Therapy. 20 (9): 1737–1749. doi:10.1038/mt.2012.119. PMC 3437581. PMID 22735378. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3437581" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3437581</a> <a href="#fnref:10" class="footnote-back-ref">↩</a></p></li> <li id="fn:11"><p>Gu, S.; Jin, L.; Zhang, Y.; Huang, Y.; Zhang, F.; Valdmanis, P. N.; Kay, M. A. (2012). "The Loop Position of shRNAs and Pre-miRNAs is Critical for the Accuracy of Dicer Processing In Vivo". Cell. 151 (4): 900–911. doi:10.1016/j.cell.2012.09.042. PMC 3499986. PMID 23141545. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499986" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499986</a> <a href="#fnref:11" class="footnote-back-ref">↩</a></p></li> <li id="fn:12"><p>Suhy, D. A.; Kao, S. C.; Mao, T.; Whiteley, L.; Denise, H.; Souberbielle, B.; Burdick, A. D.; Hayes, K.; Wright, J. F.; Lavender, H.; Roelvink, P.; Kolykhalov, A.; Brady, K.; Moschos, S. A.; Hauck, B.; Zelenaia, O.; Zhou, S.; Scribner, C.; High, K. A.; Renison, S. H.; Corbau, R. (2012). "Safe, Long-term Hepatic Expression of Anti-HCV shRNA in a Nonhuman Primate Model". Molecular Therapy. 20 (9): 1737–1749. doi:10.1038/mt.2012.119. PMC 3437581. PMID 22735378. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3437581" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3437581</a> <a href="#fnref:12" class="footnote-back-ref">↩</a></p></li> <li id="fn:13"><p>Suhy, D. A.; Kao, S. C.; Mao, T.; Whiteley, L.; Denise, H.; Souberbielle, B.; Burdick, A. D.; Hayes, K.; Wright, J. F.; Lavender, H.; Roelvink, P.; Kolykhalov, A.; Brady, K.; Moschos, S. A.; Hauck, B.; Zelenaia, O.; Zhou, S.; Scribner, C.; High, K. A.; Renison, S. H.; Corbau, R. (2012). "Safe, Long-term Hepatic Expression of Anti-HCV shRNA in a Nonhuman Primate Model". Molecular Therapy. 20 (9): 1737–1749. doi:10.1038/mt.2012.119. PMC 3437581. 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