Rapid, cell-based toxicity screen of potentially therapeutic post-transcriptional gene silencing agents.
Post-transcriptional gene silencing (PTGS) agents such as antisense, ribozymes and RNA interference (RNAi) have great potential as therapeutics for a variety of eye diseases including retinal and macular degenerations, glaucoma, corneal degenerations, inflammatory and viral conditions. Despite their great potential and over thirty years of academic and corporate research only a single PTGS agent is currently approved for human therapy for a single disease. Substantial challenges exist to achieving both efficacious and safe PTGS agents. Efficacy, as measured in specific target mRNA and protein knockdown, depends upon a number of complex factors including the identification of rare regions of target mRNA accessibility, cellular co-localization of the PTGS agent in sufficient concentration with the target mRNA, and stability of the PTGS agent in the target cells in which it is delivered or expressed. Safety is commonly measured by lack of cytotoxicity or other deleterious cellular responses in cells in which the PTGS agent is delivered or expressed. To relieve major bottlenecks in RNA drug discovery novel, efficient, inexpensive, and rapid tools are needed to facilitate lead identification of the most efficacious PTGS agent, rational optimization of efficacy of the lead agent, and lead agent safety determinations. We have developed a technological platform using cell culture expression systems that permits lead identification and efficacy optimization of PTGS agents against arbitrary disease target mRNAs under relatively high throughput conditions. Here, we extend the technology platform to include PTGS safety determinations in cultured human cells that are expected to represent the common cellular housekeeping microenvironment. We developed a high throughput screening (HTS) cytotoxicity assay in 96-well plate format based around the SYTOX Green dye which is excluded from healthy viable cells and becomes substantially fluorescent only after entering cells and binding to nuclear DNA. In this format we can test a number of PTGS agents for cellular toxicity relative to control elements. We also developed an HTS 96-well plate assay that allows us to assess the impact of any given PTGS agent on stimulating a variety of common cellular stress signaling pathways (e.g. CRE, SRE, AP-1, NFκB, Myc, and NFAT) that could indicate possible deleterious effects of PTGS agents either dependent or independent of base pairing complementarity with target mRNAs. To this end we exploited the secreted alkaline phosphatase (SEAP) Pathway Profiling System where the expression of the secreted reporter protein is coupled to transcriptional activation of a variety of promoter elements involved in common cell signaling pathways. We found that a variety of lead hammerhead ribozyme (hhRz) and short hairpin (shRNA) expression constructs did not exert cytotoxicity in human cells when driven by highly active RNA Pol-III promoters. We also found that most of the cell signaling pathways tested (CRE, SRE, Myc, and NFAT) did not significantly couple through upregulation to expression of the set of PTGS agents tested. AP-1 and NFκB upregulation both appear to couple to the expression of some PTGS agents which likely reflect the known properties of these pathways to be stimulated by abundant small structured RNAs.
Department of Ophthalmology (Ross Eye Institute), University at Buffalo-SUNY, Buffalo, NY 14209, United States.
Tiffany A
TA
Kolniak
Jack M
JM
Sullivan
D005822:Genetic Vectors; D057809:HEK293 Cells / Q000187:drug effects; D057166:High-Throughput Screening Assays; D006801:Humans; D010957:Plasmids; D012313:RNA / Q000235:genetics; D034622:RNA Interference / Q000502:physiology*; D016337:RNA, Catalytic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D051117:Scavenger Receptors, Class A / Q000235:genetics; D037941:Silencer Elements, Transcriptional / Q000502:physiology; D015533:Transcriptional Activation; D014162:Transfection
C495139:MSR1 protein, human; D016337:RNA, Catalytic; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051117:Scavenger Receptors, Class A; C427204:hammerhead ribozyme; D012313:RNA
Toxicol Pathol. 1999 Jan-Feb;27(1):95-100
10367680
EMBO J. 2006 Jul 26;25(14):3335-46
16858407
Curr Eye Res. 2006 May;31(5):435-40
16714235
Biotechniques. 1998 Aug;25(2):240-4
9714883
Nat Biotechnol. 2005 Nov;23(11):1399-405
16273073
J Biol Chem. 2005 Feb 25;280(8):7010-21
15591054
Immunity. 1999 Dec;11(6):721-31
10626894
Mol Ther. 2006 Oct;14(4):463-70
16877044
Hum Mol Genet. 1997 Sep;6(9):1415-26
9285777
J Biol Chem. 2001 Feb 2;276(5):3046-53
11042199
Nature. 2006 May 25;441(7092):537-41
16724069
Am J Hum Genet. 2007 Jul;81(1):127-35
17564969
J Biol Chem. 2003 Feb 14;278(7):4416-23
12468535
Mol Vis. 2008 Jun 10;14:1075-80
18552981
Gene. 1988 Jun 15;66(1):1-10
3417148
Nat Biotechnol. 2005 Apr;23(4):457-62
15778705
Nature. 2008 Apr 3;452(7187):591-7
18368052
Mol Cell Biol. 1995 Jan;15(1):540-51
7528330
Nat Med. 1998 Aug;4(8):967-71
9701253
Gene. 1995 Aug 19;161(2):189-93
7665077
Am J Ophthalmol. 2002 Apr;133(4):552-6
11931791
Mol Cell Biol. 2000 Feb;20(4):1278-90
10648614
Genes Dev. 1989 Dec;3(12A):1991-2002
2559873
Antimicrob Agents Chemother. 1996 Sep;40(9):2004-11
8878571
Proc Natl Acad Sci U S A. 2000 Oct 10;97(21):11488-93
11005848
Exp Eye Res. 2007 Jan;84(1):44-52
17083931
Biochem Soc Trans. 2004 Dec;32(Pt 6):952-6
15506933
Oncogene. 2000 Mar 9;19(11):1369-78
10723127
Mol Cell Biol. 1985 Aug;5(8):2051-60
3018548
RNA. 1997 Jun;3(6):677-87
9174101
Nat Immunol. 2004 Jul;5(7):730-7
15208624
Exp Eye Res. 2016 Oct;151:236-55
27233447
J Biomol Screen. 2003 Jun;8(3):283-91
12857382
EMBO J. 2002 Mar 1;21(5):857-64
11867514
J Biol Chem. 2003 Sep 5;278(36):34427-37
12826675
Br J Pharmacol. 2006 Jul;148(6):833-44
16751794
Gene Ther. 2005 Aug;12(15):1223-8
15877050
Science. 2002 Apr 19;296(5567):550-3
11910072
Exp Eye Res. 2009 May;88(5):859-79
19094986
Nat Immunol. 2006 Feb;7(2):131-7
16424890
Vision Res. 2008 Feb;48(3):453-69
17976683