RNAi Shooting the Messenger!

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1 RNAi Shooting the Messenger! Bronya Keats, Ph.D. Department of Genetics Louisiana State University Health Sciences Center New Orleans

2 RNA interference (RNAi) A mechanism by which RNA suppresses gene expression Discovered in 1998 when injection of double-stranded RNA (dsrna) into C. elegans was shown to interfere with messenger RNA (mrna) containing complementary sequence Technique being used in clinical trials for age-related macular degeneration Andrew Fire and Craig Mello received Nobel Prize in 2006 for discovery of RNAi mechanism

3 m Non-coding RNA (microrna) RNAi

4 RNAi discovery was preceded by confusing outcomes with petunias in 1990

5 Introduction of additional purple transgenes resulted in white flowers! Further experiments showed that purple gene mrna was in fact reduced (not increased) by introduction of the transgenes.

6 RNAi RNAi occurs naturally in cells RNAi mechanism can be coopted to inhibit target genes not naturally silenced in specific cells

7 MicroRNA (mirna) mirna genes are transcribed, then form hairpin RNA structures, which are cut and processed to small pieces of RNA (21-23 nt) that block mrna translation to protein First described in 1993 in C. elegans but not called microrna until an extensive class of these small RNAs was discovered in 2001

8 MicroRNAs

9 Double-stranded RNA (dsrna) Non-coding dsrnas may be endogenous or introduced by viruses As with mirnas, they are cut and processed by Dicer enzymes to segments called small interfering RNAs (sirnas) Viral sirnas inhibit virus infections by destroying the viral mrna when it tries to produce proteins in the cell

10 A B C Interference with mrna occurs via small dsrna that may be (A) endogenous, (B) replicating viruses, (C) micrornas. All are cleaved by the enzyme, Dicer, into sirnas that become single-stranded and stop translation of mrna (for regulation and defense purposes in the cell).

11 RISC One strand (guide) of the sirna is selected by proteins called Argonaute, and is incorporated into RISC (RNA- Induced Silencing Complex) RISC attaches to target mrna and stops protein production (The passenger strand is degraded)

12

13 Co-opting the natural RNAi pathway ds sirnas or short hairpin RNAs (shrnas) resembling mirnas are created ds sirnas are inserted directly into cells where they are loaded into RISC in the cytoplasm shrnas use a viral vector (e.g. AAV) and are initially processed in the nucleus mimicking mirna

14 Selecting the sirna sequence

15 RNA reminder table RNAi - RNA interference mrna - messenger RNA dsrna - double-stranded RNA mirna - microrna sirna - small interfering RNA shrna - short hairpin RNA

16 Dominant Disorders One normal and one disease allele Disease allele must be silenced Design and deliver shrna that degrades the disease sequence Test in mouse models

17 Neurodegenerative Disorders

18 SCA1 Silence the expression of the CAG expansion gene In 2004 Beverly Davidson and colleagues reduced symptoms of SCA1 in a mouse model using RNAi gene therapy In cerebellar Purkinje cells, this mouse has the human transgene with 82 CAG repeats as well as the two normal mouse genes This mouse has progressive ataxia and nuclear inclusion bodies with high levels of mutant ataxin-1 in Purkinje cells

19 Approach and Results for SCA1 An efficient SCA1 shrna was selected and AAVshSCA1 was injected into the cerebellar lobules of 7 week old mice Significant improvements in motor function were seen by 11 to 21 weeks of age Long-term expression of shrna did not have any obvious detrimental effects Levels of human ataxin-1 and nuclear inclusions were reduced

20 Rotarod performance

21 RNAi reduces intranuclear inclusions

22 RNAi Issues Most effective inhibitory sequences need to be determined and designed Does sirna interfere with non-target mrna? Toxicity concerns Delivery to brain? Direct delivery gives transient effect and must be continual (via pump) or repeated for long-term studies Viral-mediated delivery provides long-term expression, but unknown adverse effects Transvascular delivery of sirna to the brain using RVG-coated nanoparticles

23 A therapy for SCA1? shsca1 inhibits production of both normal and mutant human ataxin-1 However, the ataxin-1 knockout mouse has minimal ataxia and cerebellar pathology, which may mean that reducing levels of both normal and mutant ataxin-1 in patients could lead to improvement

24 Other RNAi possibilities Suppress RBM17, a protein that interacts with Ataxin1 and prefers the expanded form Explore similar therapeutic approaches for other dominant ataxias Use RNAi to develop a mouse model with reduced levels of frataxin for FA studies

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