Use of Therapeutic RNAs to Accelerate Wound Healing in Diabetic Rabbit Wounds.

Introduction: Diabetes mellitus (DM) affects over 422 million people globally. Patients with DM are subject to a myriad of complications, of which diabetic foot ulcers (DFUs) are the most common with ∼25% chance of developing these wounds throughout their lifetime. Innovation: Currently there are no therapeutic RNAs approved for use in DFUs. Use of dressings containing novel layer-by-layer (LbL)-formulated therapeutic RNAs that inhibit PHD2 and miR-210 can significantly improve diabetic wound healing. These dressings provide sustained release of therapeutic RNAs to the wounds locally without systemic side effects. Clinical Problem Addressed: Diabetic foot wounds are difficult to heal and often result in significant patient morbidity and mortality. Materials and Methods: We used the diabetic neuroischemic rabbit model of impaired wound healing. Diabetes was induced in the rabbits with alloxan, and neuroischemia was induced by ligating the central neurovascular bundle of each ear. Four 6-mm full-thickness wounds were created on each ear. A LbL technique was used to conformally coat the wound dressings with chemically modified RNAs, including an antisense oligonucleotide (antimiR) targeting microRNA-210 (miR-210), an short synthetic hairpin RNA (sshRNA) targeting PHD2, or both. Results: Wound healing was improved by the antimiR-210 but not the PHD2-sshRNA. Specific knockdown of miR-210 in tissue as measured by RT-qPCR was ∼8 Ct greater than nonspecific controls, and this apparent level of knockdown (>99%) suggests that delivery to the tissue is highly efficient at the administered dose. Discussion: Healing of ischemic/neuropathic wounds in diabetic rabbits was accelerated upon inhibition of miR-210 by LbL delivery to the wound bed. miR-210 inhibition was achieved using a chemically modified antisense RNA.

Correction: Chhalliyil et al. A Real-Time Quantitative PCR Method Specific for Detection and Quantification of the First Commercialized Genome-Edited Plant. Foods 2020, 9, 1245.

The stated objective of the paper was to provide regulatory laboratories and industry laboratories a complete, legally robust method for the quantitative detection of SU canola [...].

A Real-Time Quantitative PCR Method Specific for Detection and Quantification of the First Commercialized Genome-Edited Plant.

Discussion regarding the regulatory status of genome-edited crops has focused on precision of editing and on doubts regarding the feasibility of analytical monitoring compliant with existing GMO regulations. Effective detection methods are important, both for regulatory enforcement and traceability in case of biosafety, environmental or socio-economic impacts. Here, we approach the analysis question for the first time in the laboratory and report the successful development of a quantitative PCR detection method for the first commercialized genome-edited crop, a canola with a single base pair edit conferring herbicide tolerance. The method is highly sensitive and specific (quantification limit, 0.05%), compatible with the standards of practice, equipment and expertise typical in GMO laboratories, and readily integrable into their analytical workflows, including use of the matrix approach. The method, validated by an independent laboratory, meets all legal requirements for GMO analytical methods in jurisdictions such as the EU, is consistent with ISO17025 accreditation standards and has been placed in the public domain. Having developed a qPCR method for the most challenging class of genome edits, single-nucleotide variants, this research suggests that qPCR-based method development may be applicable to virtually any genome-edited organism. This advance resolves doubts regarding the feasibility of extending the regulatory approach currently employed for recombinant DNA-based GMOs to genome-edited organisms.

Acceleration of Diabetic Wound Healing with PHD2- and miR-210-Targeting Oligonucleotides.

In diabetes-associated chronic wounds, the normal response to hypoxia is impaired and many cellular processes involved in wound healing are hindered. Central to the hypoxia response is hypoxia-inducible factor-1α (HIF-1α), which activates multiple factors that enhance wound healing by promoting cellular motility and proliferation, new vessel formation, and re-epithelialization. Prolyl hydroxylase domain-containing protein 2 (PHD2) regulates HIF-1α activity by targeting it for degradation under normoxia. HIF-1α also upregulates microRNA miR-210, which in turn regulates proteins involved in cell cycle control, DNA repair, and mitochondrial respiration in ways that are antagonistic to wound repair. We have identified a highly potent short synthetic hairpin RNA (sshRNA) that inhibits expression of PHD2 and an antisense oligonucleotide (antimiR) that inhibits miR-210. Both oligonucleotides were chemically modified for improved biostability and to mitigate potential immunostimulatory effects. Using the sshRNA to silence PHD2 transcripts stabilizes HIF-1α and, in combination with the antimiR targeting miR-210, increases proliferation and migration of keratinocytes in vitro. To assess activity and delivery in an impaired wound healing model in diabetic mice, PHD2-targeting sshRNAs and miR-210 antimiRs both alone and in combination were formulated for local delivery to wounds using layer-by-layer (LbL) technology. LbL nanofabrication was applied to incorporate sshRNA into a thin polymer coating on a Tegaderm mesh. This coating gradually degrades under physiological conditions, releasing sshRNA and antimiR for sustained cellular uptake. Formulated treatments were applied directly to splinted full-thickness excisional wounds in db/db mice. Cellular uptake was confirmed using fluorescent sshRNA. Wounds treated with a single application of PHD2 sshRNA or antimiR-210 closed 4 days faster than untreated wounds, and wounds treated with both oligonucleotides closed on average 4.75 days faster. Markers for neovascularization and cell proliferation (CD31 and Ki67, respectively) were increased in the wound area following treatment, and vascular endothelial growth factor (VEGF) was increased in sshRNA-treated wounds. Our results suggest that silencing of PHD2 and miR-210 either together or separately by localized delivery of sshRNAs and antimiRs is a promising approach for the treatment of chronic wounds, with the potential for rapid clinical translation.

Inhibition of hepatitis C virus in chimeric mice by short synthetic hairpin RNAs: sequence analysis of surviving virus shows added selective pressure of combination therapy.

UNLABELLED: We have recently shown that a cocktail of two short synthetic hairpin RNAs (sshRNAs), targeting the internal ribosome entry site of hepatitis C virus (HCV) formulated with lipid nanoparticles, was able to suppress viral replication in chimeric mice infected with HCV GT1a by up to 2.5 log10 (H. Ma et al., Gastroenterology 146:63-66.e5, http://dx.doi.org/10.1053/j.gastro.2013.09.049) Viral load remained about 1 log10 below pretreatment levels 21 days after the end of dosing. We have now sequenced the HCV viral RNA amplified from serum of treated mice after the 21-day follow-up period. Viral RNA from the HCV sshRNA-treated groups was altered in sequences complementary to the sshRNAs and nowhere else in the 500-nucleotide sequenced region, while the viruses from the control group that received an irrelevant sshRNA had no mutations in that region. The ability of the most commonly selected mutations to confer resistance to the sshRNAs was confirmed in vitro by introducing those mutations into HCV-luciferase reporters. The mutations most frequently selected by sshRNA treatment within the sshRNA target sequence occurred at the most polymorphic residues, as identified from an analysis of available clinical isolates. These results demonstrate a direct antiviral activity with effective HCV suppression, demonstrate the added selective pressure of combination therapy, and confirm an RNA interference (RNAi) mechanism of action. IMPORTANCE: This study presents a detailed analysis of the impact of treating a hepatitis C virus (HCV)-infected animal with synthetic hairpin-shaped RNAs that can degrade the virus's RNA genome. These RNAs can reduce the viral load in these animals by over 99% after 1 to 2 injections. The study results confirm that the viral rebound that often occurred a few weeks after treatment is due to emergence of a virus whose genome is mutated in the sequences targeted by the RNAs. The use of two RNA inhibitors, which is more effective than use of either one by itself, requires that any resistant virus have mutations in the targets sites of both agents, a higher hurdle, if the virus is to retain the ability to replicate efficiently. These results demonstrate a direct antiviral activity with effective HCV suppression, demonstrate the added selective pressure of combination therapy, and confirm an RNAi mechanism of action.

Formulated minimal-length synthetic small hairpin RNAs are potent inhibitors of hepatitis C virus in mice with humanized livers.

Short synthetic hairpin RNAs (sshRNAs) (SG220 and SG273) that target the internal ribosome entry site of the hepatitis C virus (HCV) were formulated into lipid nanoparticles and administered intravenously to HCV-infected urokinase plasminogen activator-severe combined immunodeficient mice with livers repopulated with human hepatocytes (humanized livers). Weekly administration of 2.5 mg/kg of each sshRNA for 2 weeks resulted in a maximal mean reduction in viral load of 2.5 log10 from baseline. The viral load remained reduced by more than 90% at 14 days after the last dose was given. The sshRNAs were well tolerated and did not significantly increase liver enzyme levels. These findings indicate the in vivo efficacy of a synthetic RNA inhibitor against the HCV genome in reducing HCV infection.

Minimal-length Synthetic shRNAs Formulated with Lipid Nanoparticles are Potent Inhibitors of Hepatitis C Virus IRES-linked Gene Expression in Mice.

We previously identified short synthetic shRNAs (sshRNAs) that target a conserved hepatitis C virus (HCV) sequence within the internal ribosome entry site (IRES) of HCV and potently inhibit HCV IRES-linked gene expression. To assess in vivo liver delivery and activity, the HCV-directed sshRNA SG220 was formulated into lipid nanoparticles (LNP) and injected i.v. into mice whose livers supported stable HCV IRES-luciferase expression from a liver-specific promoter. After a single injection, RNase protection assays for the sshRNA and (3)H labeling of a lipid component of the nanoparticles showed efficient liver uptake of both components and long-lasting survival of a significant fraction of the sshRNA in the liver. In vivo imaging showed a dose-dependent inhibition of luciferase expression (>90% 1 day after injection of 2.5 mg/kg sshRNA) with t1/2 for recovery of about 3 weeks. These results demonstrate the ability of moderate levels of i.v.-injected, LNP-formulated sshRNAs to be taken up by liver hepatocytes at a level sufficient to substantially suppress gene expression. Suppression is rapid and durable, suggesting that sshRNAs may have promise as therapeutic agents for liver indications.Molecular Therapy-Nucleic Acids (2013) 2, e123; doi:10.1038/mtna.2013.50; published online 17 September 2013.

Right- and left-loop short shRNAs have distinct and unusual mechanisms of gene silencing.

Small hairpin RNAs (shRNAs) having duplex lengths of 25-29 bp are normally processed by Dicer into short interfering RNAs (siRNAs) before incorporation into the RNA-induced silencing complex (RISC). However, shRNAs of ≤ 19 bp [short shRNAs (sshRNAs)] are too short for Dicer to excise their loops, raising questions about their mechanism of action. sshRNAs are designated as L-type or R-type according to whether the loop is positioned 3' or 5' to the guide sequence, respectively. Using nucleotide modifications that inhibit RNA cleavage, we show that R- but not L-sshRNAs require loop cleavage for optimum activity. Passenger-arm slicing was found to be important for optimal functioning of L-sshRNAs but much less important for R-sshRNAs that have a cleavable loop. R-sshRNAs could be immunoprecipitated by antibodies to Argonaute-1 (Ago1); complexes with Ago1 contained both intact and loop-cleaved sshRNAs. In contrast, L-sshRNAs were immunoprecipitated with either Ago1 or Ago2 and were predominantly sliced in the passenger arm of the hairpin. However, 'pre-sliced' L-sshRNAs were inactive. We conclude that active L-sshRNAs depend on slicing of the passenger arm to facilitate opening of the duplex, whereas R-sshRNAs primarily act via loop cleavage to generate a 5'-phosphate at the 5'-end of the guide strand.

Effects of chemical modification on the potency, serum stability, and immunostimulatory properties of short shRNAs.

Small hairpin RNAs (shRNAs) with 19-base-pair, or shorter, stems (short shRNAs [sshRNAs]) have been found to constitute a class whose mechanism of action appears to be distinct from that of small interfering RNAs (siRNAs) or longer shRNAs. These sshRNAs can be as active as canonical siRNAs or longer shRNAs. Their activity is affected by whether the antisense strand is positioned 5' or 3' to the loop (L or R sshRNAs, respectively). Dicer seems not to be involved in the processing of sshRNAs, although the mechanism of target gene suppression by these hairpins is through Ago2-mediated mRNA cleavage. In this study, the effects of chemical modifications on the potency, serum stability, and innate immune response of sshRNAs were investigated. Deoxynucleotide substitution and 2'-O-methyl (2'-OMe) modification in the sense strand and loop did not affect silencing activity, but, unlike with siRNAs, when placed in the antisense strand these modifications were detrimental. Conjugation with bulky groups at the 5'-end of L sshRNAs or 3'-end of R sshRNAs had a negative impact on the potency. Unmodified sshRNAs in dimer form or with blunt ends were immunostimulatory. Some modifications such as 3'-end conjugation and phosphorothioate linkages on the backbone of the sshRNAs could also induce inflammatory cytokine production. However, 2'-OMe substitution of sshRNAs abrogated the innate immune response and improved the serum stability of the hairpins.

Minimal-length short hairpin RNAs: the relationship of structure and RNAi activity.

Small hairpin RNAs (shRNAs) are widely used in RNAi studies and typically consist of a stem of 19-29 base pairs (bp), a loop of at least 4 nucleotides (nt), and a dinucleotide overhang at the 3' end. Compared with shRNAs with 21-29 bp stems, we have found that shRNAs with 19-bp or shorter stems (sshRNAs) possess some unique structure-activity features that depend on whether the antisense strand is positioned 5' or 3' to the loop (L- or R-type sshRNAs, respectively). L sshRNAs can have IC(50)s in the very low picomolar range, and sshRNAs with nominal loop sizes of 1 or 4 nt were at least as active as those with longer loops. L sshRNAs remained highly potent even when the 3' end of the antisense strand was directly linked with the 5' end of the sense strand. In this case, the sense strand can be shorter than the antisense strand, and the loop can be formed entirely by the 3' end of the antisense strand. Monomer sshRNAs are not processed by recombinant Dicers in vitro. Although they can form dimers that are sometimes Dicer substrates, their RNAi activity is not dependent on the formation of such structures. Our findings have implications for the mechanism of action of sshRNAs, and the ability to design highly potent shRNAs with minimal length is encouraging for the prospects of the therapeutic use of direct-delivered shRNAs.

Stability study of unmodified siRNA and relevance to clinical use.

RNA interference offers enormous potential to develop therapeutic agents for a variety of diseases. To assess the stability of siRNAs under conditions relevant to clinical use with particular emphasis on topical delivery considerations, a study of three different unmodified siRNAs was performed. The results indicate that neither repeated freeze/thaw cycles, extended incubations (over 1 year at 21 degrees C), nor shorter incubations at high temperatures (up to 95 degrees C) have any effect on siRNA integrity as measured by nondenaturing polyacrylamide gel electrophoresis and functional activity assays. Degradation was also not observed following exposure to hair or skin at 37 degrees C. However, incubation in fetal bovine or human sera at 37 degrees C led to degradation and loss of activity. Therefore, siRNA in the bloodstream is likely inactivated, thereby limiting systemic exposure. Interestingly, partial degradation (observed by gel electrophoresis) did not always correlate with loss of activity, suggesting that partially degraded siRNAs retain full functional activity. To demonstrate the functional activity of unmodified siRNA, EGFP-specific inhibitors were injected into footpads and shown to inhibit preexisting EGFP expression in a transgenic reporter mouse model. Taken together, these data indicate that unmodified siRNAs are viable therapeutic candidates.

Hairpin ribozyme-antisense RNA constructs can act as molecular Lassos.

We have developed a novel class of antisense agents, RNA Lassos, which are capable of binding to and circularizing around complementary target RNAs. The RNA Lasso consists of a fixed sequence derived from the hairpin ribozyme and an antisense segment whose size and sequence can be varied to base pair with accessible sites in the target RNA. The ribozyme catalyzes self-processing of the 5'- and 3'-ends of a transcribed Lasso precursor and ligates the processed ends to produce a circular RNA. The circular and linear forms of the self-processed Lasso coexist in an equilibrium that is dependent on both the Lasso sequence and the solution conditions. Lassos form strong, noncovalent complexes with linear target RNAs and form true topological linkages with circular targets. Lasso complexes with linear RNA targets were detected by denaturing gel electrophoresis and were found to be more stable than ordinary RNA duplexes. We show that expression of a fusion mRNA consisting of a sequence from the murine tumor necrosis factor-alpha (TNF-alpha) gene linked to luciferase reporter can be specifically and efficiently blocked by an anti-TNF Lasso. We also show in cell culture experiments that Lassos directed against Fas pre-mRNA were able to induce a change in alternative splicing patterns.

shRNAs targeting hepatitis C: effects of sequence and structural features, and comparision with siRNA.

Hepatitis C virus (HCV) is a leading cause of liver cirrhosis and hepatocellular carcinoma worldwide. Currently available treatment options are of limited efficacy, and there is an urgent need for development of alternative therapies. RNA interference (RNAi) is a natural mechanism by which small interfering RNA (siRNA) or short hairpin RNA (shRNA) can mediate degradation of a target RNA molecule in a sequence-specific manner. In this study, we screened in vitro-transcribed 25-bp shRNAs targeting the internal ribosome entry site (IRES) of HCV for the ability to inhibit IRES-driven gene expression in cultured cells. We identified a 44-nt region at the 3'-end of the IRES within which all shRNAs efficiently inhibited expression of an IRES-linked reporter gene. Subsequent scans within this region with 19-bp shRNAs identified even more potent molecules, providing effective inhibition at concentrations of 0.1 nM. Experiments varying features of the shRNA design showed that, for 25-bp shRNAs, neither the size of the loop (4-10 nt) nor the sequence or pairing status of the ends affects activity, whereas in the case of 19-bp shRNAs, larger loops and the presence of a 3'-UU overhang increase efficacy. A comparison of shRNAs and siRNAs targeting the same sequence revealed that shRNAs were of comparable or greater potency than the corresponding siRNAs. Anti-HCV activity was confirmed with HCV subgenomic replicons in a human hepatocyte line. The results indicate that shRNAs, which can be prepared by either transcription or chemical synthesis, may be effective agents for the control of HCV.

Delivery and inhibition of reporter genes by small interfering RNAs in a mouse skin model.

RNA interference offers the potential of a novel therapeutic approach for treating skin disorders. To this end, we investigated delivery of nucleic acids, including a plasmid expressing the reporter gene luciferase, to mouse skin by intradermal injection into footpads using in vivo bioluminescence imaging over multiple time points. In order to evaluate the ability of RNA interference to inhibit skin gene expression, reporter gene constructs were co-injected with specific or non-specific siRNAs and the in vivo effects measured. Our results revealed that specific unmodified and modified siRNAs (but not nonspecific matched controls) strongly inhibit reporter gene expression in mice. These results indicate that small interfering RNA, delivered locally as RNA directly or expressed from viral or non-viral vectors, may be effective agents for treating skin disorders.

Inhibition of hepatitis C IRES-mediated gene expression by small hairpin RNAs in human hepatocytes and mice.

The ability of small hairpin RNAs (shRNAs) to inhibit hepatitis C virus internal ribosome entry site (HCV IRES)-dependent gene expression was investigated in cultured cells and a mouse model. The results indicate that shRNAs, delivered as naked RNA or expressed from vectors, may be effective agents for the control of HCV and related viruses.

Small hairpin RNAs efficiently inhibit hepatitis C IRES-mediated gene expression in human tissue culture cells and a mouse model.

Treatment and prevention of hepatitis C virus (HCV) infections remain a major challenge for controlling this worldwide health problem; existing therapies are only partially effective and no vaccine is currently available. RNA interference offers the potential of a novel therapeutic approach for treating HCV infections. Toward this end, we evaluated small hairpin interfering RNAs (shRNAs) targeting the conserved internal ribosome entry site (IRES) element of the HCV genome for their ability to control gene expression in human cells and animals. We used a reporter gene plasmid in which firefly luciferase (fLuc) expression is dependent on the HCV IRES. Direct delivery of HCV IRES shRNAs efficiently blocked HCV IRES-mediated fLuc expression in transfected human 293FT cells as well as in a mouse model in which nucleic acids were delivered to liver cells by hydrodynamic transfection via the tail vein. These results indicate that shRNAs, delivered as RNA or expressed from viral or nonviral vectors, may be effective agents for the control of HCV and related viruses.

Complete, gene-specific siRNA libraries: production and expression in mammalian cells.

Short interfering RNAs (siRNAs) are widely used to silence the expression of specific genes. Current practice for designing effective siRNAs is to use algorithms based on sequence-efficacy correlations; however, there are many highly effective sequences that these algorithms do not anticipate. To ensure that the best siRNAs are identified, all possible gene-specific siRNA sequences of appropriate lengths should be screened in cell culture. Synthesizing and testing all such sequences individually is costly. A potentially much easier alternative is to prepare a mixture of all these sequences (a gene-specific library), express them in cells, select cells having the desired phenotype, and identify the siRNA contained within the selected cells. Here we describe two new methods for preparing and expressing such libraries. The first uses cloned Dicer or RNase III to digest gene-specific RNA duplexes to siRNAs, which are then converted to the corresponding DNA sequences by attaching RNA primers and performing reverse transcription-PCR. The second method involves partial DNase I digestion of gene-specific DNA, purification of a 20-30-bp fraction, and amplification by attaching DNA adapters followed by PCR. DNA libraries specific for TNF-alpha, DsRed, and part of the hepatitis C virus genome, generated by methods, were inserted into siRNA expression vectors between convergent human U6 and H1 promoters. Randomly selected clones from each library together with vectors expressing the corresponding target genes were cotransfected into 293FT cells and assayed for target gene inhibition. About 10%-20% of siRNAs represented in these libraries show significant inhibition of their target genes. Most of these inhibitory sequences are not predicted by existing algorithms.