697 New Insights Into the Mechanisms of Argonaute Protein Competition and Implications for RNAi Gene Therapies Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of G[.]
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directly to cRGD peptide or to internalizing RGD (iRGD), which
is a variant RGD peptide that triggers permeabilization of tumor
endothelium and internalization by cells through secondary binding
to neuropilin The cRGD- and iRGD-conjugated KRAS Adaptors
were tested for efficacy against subcutaneous MiaPaca-2 xenografts,
and tumor growth was inhibited to equal or greater extent as with
the original cRGD-dendrimer system iRGD may be of particular
benefit for pancreatic adenocarcinomas, which have a densely fibrotic
stroma that impedes drug delivery Enhanced delivery of small- and
large-molecule therapeutics into primary pancreatic adenocarcinomas
in KPC mice has been achieved previously by conjugating or
co-injecting iRGD peptide
We have shown that U1 Adaptors can successfully target human
KRAS both in vitro and in vivo These results support the continued
development of U1 Adaptor technology as a strategy for therapeutic
suppression of KRAS in pancreatic cancer
695 Splice-Correction of X-Linked
Agammaglobulinemia in a Human BAC-Transgenic
Mouse Model Using Oligonucleotides
Burcu Bestas,1 Pedro M.D Moreno,1 Emelie K.M Blomberg,1
Dara K Mohammad,1 Karin E Lundin,1 Robert Månsson,2 Anna
Berglöf,1 Jesper Wengel,3 Edvard C.I Smith.1
1 Laborattory Medicine, Karolinska Institutet, Huddinge/Stockholm,
Sweden; 2 Medicine, Karolinska Institutet, Huddinge/Stockholm,
Sweden; 3 Physics Chemistry and Pharmacy, University of Southern
Denmark, Odense, Denmark.
The inherited immunodeficiency, X-linked agammaglobulinemia
(XLA), is caused by mutations in the BTK gene, and results in
a B-lineage developmental block We have recently assessed the
treatment potential of splice-correcting oligonucleotides (SCOs)
targeting a mutated BTK transcript, which contains a pseudo-exon
(Bestas et al., J Clin Invest 124: 4067, 2014) In order to study
the potential of SCOs, we engineered a novel, Bacterial Artificial
Chromosome (BAC)-transgenic mouse carrying a mutated human
BTK gene, originally found in an XLA family In order to avoid
any influence of mouse endogenous Btk protein, we bred the
BAC-transgenic mice onto a Btk knockout background In this model it
was possible to correct the defect both in pro-B-cells in vitro, and
also in mature B-cells, as demonstrated by the injection of SCOs in
vivo The corrected mRNA gave rise to a functional BTK protein
As a final proof-of-concept we were also able to correct the defect
in primary patient cells In this study we used different nucleotide
chemistries, 2’-O-methyl, locked nucleic acid (LNA) and morpholino
chemistries We have now included also other nucleotide chemistries
in order to make comparative studies This is to our knowledge the
first time that a lymphocyte defect, caused by abnormal splicing, has
been corrected in vivo using a splice-correction approach
696 Pre-Clinical Evaluation of Allele-Specific
Mutant Huntingtin Gene Silencing Antisense
Oligonucleotides
Amber L Southwell,1 Niels H Skotte,1 Nicholas Caron,1 Holly
Kordasiewicz,2 Michael Oestergaard,2 Crystal N Doty,1 Erika B
Villanueva,1 Yuanyun Xie,1 Boguslaw Felczak,1 Susan M Freier,2
Eric E Swayze,2 Punit P Seth,2 C Frank Bennet,2 Michael R
Hayden.1
1 Centre for Molecular Medicine and Therapeutics, University of
British Columbia, Vancouver, BC, Canada; 2 ISIS Pharmaceuticals,
Carlsbad, CA.
Huntington disease (HD) is a dominant, genetic neurodegenerative
disease characterized by progressive loss of voluntary motor
control, psychiatric disturbance, and cognitive decline, for which
there is currently no disease-modifying therapy HD is caused by the expansion of a CAG tract in the huntingtin (HTT) gene The mutant HTT protein (muHTT) acquires toxic functions, and there is significant evidence that muHTT lowering would be therapeutically efficacious However, the wild-type HTT protein (wtHTT) serves vital functions, making allele-specific muHTT lowering strategies potentially safer than non-selective strategies CAG tract expansion is associated with single nucleotide polymorphisms (SNPs) that can be targeted by gene silencing reagents such as antisense oligonucleotides (ASOs) to accomplish allele-specific muHTT lowering We have evaluated ASOs targeted to HD-associated SNPs in acute in vivo studies including screening, distribution, duration of action and dosing, using a humanized mouse model of HD, Hu97/18, that is heterozygous for the targeted SNPs We have identified four well-tolerated lead ASOs that potently and selectively silence muHTT at
a broad range of doses throughout the central nervous system for 36 weeks or more after a single intracerebroventricular injection
We next conducted a pre-clinical therapeutic efficacy trial of lead ASOs and evaluated them for effect on the HD-like phenotypes
of Hu97/18 mice Treated mice underwent longitudinal behavioral and biochemical assessment followed by terminal neuropathology Thus far we have determined that pre-symptomatic allele-specific muHTT silencing prevents onset of behavioral HD-like phenotypes Evaluation of neuropathology and post-symptomatic intervention is ongoing Contingent on findings from these studies and using delivery and dosing information gained from ongoing CNS ASO clinical trials,
a primary SNP-targeted ASO drug could be fairly rapidly translated for human applications
697 New Insights Into the Mechanisms of Argonaute Protein Competition and Implications for RNAi Gene Therapies
Mario Lederle,1 Kathrin Tegeler,1 Daniela Cerny,1 Patrick Zessin,1 Dirk Grimm.1
1 Department of Infectious Diseases/Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, Germany.
RNA interference (RNAi) has become an established tool for basic gene annotation and a promising option for therapeutic concepts that require specific inhibition of dysregulated or exogenous genes However, we and others accumulated strong evidence that over-expression of short hairpin RNAs (shRNAs, an expressable and widely used form of RNAi trigger) can cause cytotoxicity leading up
to organ damage and lethality in shRNA-treated animals A meticulous dissection of the underlying mechanisms is critical as it will ultimately guide the design of safer RNAi therapies and thus foster their clinical translation One informative finding was that co-expression of Ago2 (Slicer, as it cleaves targeted mRNA), a key component of the RNA-induced silencing complex RISC, increases shRNA efficiency in cells and animals, implying that Ago2 is a rate-limiting factor whose saturation may be involved in cytotoxicity Moreover, over-expression
of the three other mammalian Ago proteins - Ago1, Ago3 or Ago4 (all slicing-incompetent) - dampens RNAi potency, underscoring that a delicate balance of all four Ago variants is key to efficient and safe gene silencing Here, we investigated three hypotheses that could explain how relative Ago1-4 levels may affect the functionality of shRNA-loaded RISC and hence the strength of target knockdown: over-abundance of non-Slicer Ago proteins could (i) dysregulate Ago2 expression (transcription and/or translation), (ii) re-localize intra-cellular Ago2 away from sites of RNAi activity, or (iii) quantitatively sequester shRNAs into slicing-incompetent RISC Using codon-optimized Ago variants that are distinguishable by real-time PCR from the endogenous counterparts, as well as Western blotting,
we could eliminate model (i) Likewise, wide-field and confocal
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microscopy analyses revealed no evidence for Ago2 re-localization
upon Ago1/3/4 over-expression and vice versa We therefore focused
on hypothesis (iii) and fused the Ago1-4 cDNAs with GFP or RFP
tags that enabled their specific immunoprecipitation, alone or in
combination, using the TRAP system Indeed, we found that Slicer
and non-Slicers compete for shRNA loading and mRNA knockdown
in a dose-dependent manner, readily explaining all prior observations
of RNAi enhancement by Ago2 over-expression, or shRNA inhibition
by Ago1/3/4, respectively Importantly, the combination of GFP/
RFP-Ago fusion constructs with the co-precipitation protocols newly
established during this work should be widely useful to address other
vital questions in the RNAi field, such as the selective processing of
different cellular small RNAs by the four Ago proteins Moreover,
our notion that over-expression of non-Slicers has no adverse effect
on endogenous Ago2 levels or localization paves the way for novel
strategies to fine-tune therapeutic RNAi in a safe and potent manner
As a prototype, we present a new shRNA/Ago co-expression system
whereby Ago1/2 levels can be regulated independently through
exogenous macrolides, permitting a tight control of target knockdown
efficiencies and providing an original avenue to reduce toxicity in
future RNAi gene therapy applications
698 Eliminating TLR9+ Prostate Cancer Stem
Cells In Vivo Using NF-κB/RELA- or
STAT3-Targeting CpG-siRNA Conjugates
Dayson F Moreira,1 Qifang Zhang,1 Dewan M Hossain,1
Sergey Nechaev,1 Haiqing Li,1 Claudia M Kowolik,1 Massimo
D’Apuzzo,3 Stephen Forman,1 Jeremy Jones,1 Sumanta K Pal,1
Marcin Kortylewski1.1
1 Beckman Research Institute at City of Hope, Duarte, CA.
Treatment of hormone-refractory and frequently reoccurring
prostate cancers is a major clinical challenge Here, we demonstrate
that Toll-like Receptor 9 (TLR9) is commonly upregulated in
late-stage prostate cancers and provides a potential therapeutic target As
a sensor of immunogenic cell death, TLR9 bridges intra-prostatic
inflammation to cancer stem cell phenotype Our limited dilution/
serial transplantation experiments in vivo demonstrate that TLR9
is essential for prostate cancer cells’ potential to propagate and
self-renew In addition, low expression or silencing of TLR9 limits
the clonogenic potential and mesenchymal stem cell-like properties
of prostate cancer cells Genome-wide transcriptional analysis
of cancer cells isolated from xenotransplanted prostate tumors
revealed a unique TLR9-dependent gene expression signature
Further analysis of the TLR9 downstream signaling indicated that
tumorigenic transcription factors NF-κB/RELA and STAT3 cooperate
to orchestrate expression of stem cell-related genes Both RELA and
STAT3 bound and co-regulated promoters of NKX3.1 and KLF4
prostate cancer stem cell-related genes We further demonstrated the
feasibility of targeting prostate cancer-propagating potential in vivo
by TLR9-targeted siRNA delivery using CpG-siRNA conjugates
Local administration of CpG-RELAsiRNA or CpG-STAT3siRNA
but not control conjugates, inhibited tumor growth and cancer cell
clonogenic potential in two xenotransplanted prostate cancer models
Selective elimination of tumor-propagating cells using TLR9-targeted
blockade of NF-κB/RELA and STAT3 signaling has potential for
clinical translation to benefit patients with late-stage prostate cancers
699 shRNA Sense Strand Neutralization Reduces Off-Targeting, Ameliorates Toxicity and Enhances Efficacy of RNAi-Based HBV Gene Therapy
Thomas Michler,1 Stefanie Grosse,2 Stefan Mockenhaupt,2 Ulrike Protzer,1 Dirk Grimm.2
1 Virology, Technical University, Munich, Germany; 2 Virology, University of Heidelberg, Heidelberg, Germany.
A decade after first reports of successful inhibition of hepatitis B virus (HBV) replication in vivo using shRNAs, only a single RNAi therapeutic is under phase II efficacy investigation in Hepatitis
B patients This slow clinical translation is puzzling, as current standard-of-care requires life-long and daily drug treatment which is often accompanied by side effects and occurrence of resistant HBV mutants Also, while preventing the generation of infectious HBV virions, reverse-transcriptase inhibitors fail to suppress HBV surface and e-antigen, which enable viral persistence by modulating the host immune system and contribute to HBV-associated hepatocellular carcinoma These problems may be solved using RNAi as anti-HBV therapeutic since (i) long-term suppression can be achieved by a single dose of an shRNA-encoding vector, and (ii) all four HBV transcripts share a common 3 end, allowing concurrent inhibition
of HBV pre-genomic RNA and all viral proteins with a single RNAi molecule, and potentially restoring antiviral immunity and preventing carcinogenesis Still, clinical translation remains hampered
by safety concerns as shRNA over-expression in the mouse liver causes elevated liver transaminases, jaundice and weight loss One explanation is that ectopic RNAi triggers overload the endogenous miRNA pathway, perturbing miRNA biogenesis and/or activity, and causing cytotoxicity Evidence for this saturation model is that (i) shRNAs circumvent Drosha processing, a gatekeeper for miRNA biogenesis, (ii) embedding shRNAs in a recombinant miRNA context yields lower and safer levels of mature RNAi triggers, and (iii) overexpression of Argonaute-2 (Ago2) ameliorates toxicity and enhances RNAi efficacy Moreover, RNAi triggers can perturb cell physiology through unwanted inhibition of off-target genes with partial complementarities to one of the two strands of the RNAi molecule To alleviate such unintended gene silencing, we have developed a novel bi-cistronic AAV vector that expresses, in addition to the shRNA, a second RNA hairpin called tough decoy or TuD In cell culture, the TuD effectively sequestered and inactivated shRNA sense strands, and thereby improved RNAi specificity These remarkable features translated well into an HBV-transgenic mouse model, where sense strand off-target activity was likewise prevented,
as validated by transcriptome analysis of liver RNA, coinciding with ameliorated toxicity Enhanced in vivo safety was also noted for two alternative AAV vectors, either co-expressing the shRNA and Ago2,
or embedding the shRNA in a miR-122 backbone Notably, the new shRNA/TuD vector outperformed the other expression strategies regarding efficacy with stable HBV reduction of up to 98% over
3 months We speculate that its enhanced antiviral efficacy results from increased loading of the desired antisense strand into RISC in absence of the sense strand, possibly also further amending toxicity
by attenuating RISC saturation The simple TuD design and the versatility of our new AAV vector pave the way for adaptation of our strategy to other applications and should facilitate clinical translation