Impact of Phosphorothioate Chirality on Double-Stranded siRNAs: A Systematic Evaluation of Stereopure siRNA Designs.

Oligonucleotides are important therapeutic approaches, as evidenced by recent clinical successes with antisense oligonucleotides (ASOs) and double-stranded short interfering RNAs (siRNAs). Phosphorothioate (PS) modifications are a standard feature in the current generation of oligonucleotide therapeutics, but generate isomeric mixtures, leading to 2n isomers. All currently marketed therapeutic oligonucleotides (ASOs and siRNAs) are complex isomeric mixtures. Recent chemical methodologies for stereopure PS insertions have resulted in preliminary rules for ASOs, with multiple stereopure ASOs moving into clinical development. Although siRNAs have comparatively fewer PSs, the field has yet to embrace the idea of stereopure siRNAs. Herein, it has been investigated whether the individual isomers contribute equally to the in vivo activity of a representative siRNA. The results of a systematic evaluation of stereopure PS incorporation into antithrombin-3 (AT3) siRNA are reported and demonstrate that individual PS isomers dramatically affect in vivo activity. A standard siRNA design with six PS insertions was investigated and it was found that only about 10 % of the 64 possible isomers were as efficacious as the stereorandom control. Based on this data, it can be concluded that G1R stereochemistry is critical, G2R is important, G21S is preferable, and G22 and P1/P2 tolerate both isomers. Surprisingly, the disproportionate loss of efficacy for most isomers does not translate into significant gain for the productive isomers, and thus, warrants further mechanistic studies.

Induction of RNAi Responses by Short Left-Handed Hairpin RNAi Triggers.

Small double-stranded, left-handed hairpin (LHP) RNAs containing a 5'-guide-loop-passenger-3' structure induce RNAi responses by a poorly understood mechanism. To explore LHPs, we synthesized fully 2'-modified LHP RNAs targeting multiple genes and found all to induce robust RNAi responses. Deletion of the loop and nucleotides at the 5'-end of the equivalent passenger strand resulted in a smaller LHP that still induced strong RNAi responses. Surprisingly, progressive deletion of up to 10 nucleotides from the 3'-end of the guide strand resulted in a 32mer LHP capable of inducing robust RNAi responses. However, further guide strand deletion inhibited LHP activity, thereby defining the minimal length guide targeting length to 13 nucleotides. To dissect LHP processing, we examined LHP species that coimmunoprecipitated with Argonaute 2 (Ago2), the catalytic core of RNA-induced silencing complex, and found that the Ago2-associated processed LHP species was of a length that correlated with Ago2 cleavage of the passenger strand. Placement of a blocking 2'-OMe blocking modification at the LHP predicted Ago2 cleavage site resulted in an intact LHP loaded into Ago2 and no RNAi response. Taken together, these data argue that in the absence of a substantial loop, this novel class of small LHP RNAs enters the RNAi pathway by a Dicer-independent mechanism that involves Ago2 cleavage and results in an extended guide strand. This work establishes LHPs as an alternative RNAi trigger that can be produced from a single synthesis for potential use as an RNAi therapeutic.

Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi.

Gene knockout strategies, RNAi and rescue experiments are all employed to study mammalian gene function. However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels. Here, we developed an endogenous gene knockdown/rescue strategy that combines RNAi selectivity with a highly efficient CRISPR directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific mutations plus an allele-selective siRNA Sensitive (siSN) site that allows for studying gene mutations while maintaining endogenous expression and regulation of the gene of interest. CRISPR/Cas9 plus rAAV targeted gene-replacement and introduction of allele-specific RNAi sensitivity mutations in the CDK2 and CDK1 genes resulted in a >85% site-specific recombination of Neo-resistant clones versus ∼8% for rAAV alone. RNAi knockdown of wild type (WT) Cdk2 with siWT in heterozygotic knockin cells resulted in the mutant Cdk2 phenotype cell cycle arrest, whereas allele specific knockdown of mutant CDK2 with siSN resulted in a wild type phenotype. Together, these observations demonstrate the ability of CRISPR plus rAAV to efficiently recombine a genomic locus and tag it with a selective siRNA sequence that allows for allele-selective phenotypic assays of the gene of interest while it remains expressed and regulated under endogenous control mechanisms.

Efficient delivery of RNAi prodrugs containing reversible charge-neutralizing phosphotriester backbone modifications.

RNA interference (RNAi) has great potential to treat human disease. However, in vivo delivery of short interfering RNAs (siRNAs), which are negatively charged double-stranded RNA macromolecules, remains a major hurdle. Current siRNA delivery has begun to move away from large lipid and synthetic nanoparticles to more defined molecular conjugates. Here we address this issue by synthesis of short interfering ribonucleic neutrals (siRNNs) whose phosphate backbone contains neutral phosphotriester groups, allowing for delivery into cells. Once inside cells, siRNNs are converted by cytoplasmic thioesterases into native, charged phosphodiester-backbone siRNAs, which induce robust RNAi responses. siRNNs have favorable drug-like properties, including high synthetic yields, serum stability and absence of innate immune responses. Unlike siRNAs, siRNNs avidly bind serum albumin to positively influence pharmacokinetic properties. Systemic delivery of siRNNs conjugated to a hepatocyte-specific targeting domain induced extended dose-dependent in vivo RNAi responses in mice. We believe that siRNNs represent a technology that will open new avenues for development of RNAi therapeutics.

The road to therapeutic RNA interference (RNAi): Tackling the 800 pound siRNA delivery gorilla.

If those of us privileged enough to have the opportunity to work towards curing human diseases had the power to design the ideal therapeutic molecule, the question would be what selection criteria would we choose? Arguably, at the top of the list would be four mandatory properties: specificity, potency, tolerability, and universality. So it should come as no surprise the momentum associated with the field of small interfering RNA (siRNA)-induced RNA Interference (RNAi) therapeutics has gained strength, as these molecules have shown exceptional promise in fulfilling all of these requirements. Unfortunately, siRNAs are too large, too charged, and too rigid to passively diffuse across the cellular membrane and thereby require a delivery system to enter cells. Thus, since its conception of working in human cells, siRNA delivery remains The 800 Pound Gorilla in the room. The main complication yet to overcome is engineering delivery systems that are safe and efficient in systemically delivering siRNA molecules to the diseased tissue and across the cellular membrane of target cells. Currently, encapsulating the siRNA in nanoparticle and liposomal systems has risen to become the standard of delivery approaches. While generally speaking these delivery platforms offer significant advancements, our laboratory is committed to generating alternative siRNA delivery technologies that avoid nanoparticle packaging and allow siRNA molecules to be delivered as single, soluble entities. This brief review discusses the first of these technologies, a Peptide Transduction Domain-dsRNA Binding Domain (PTD-DRBD) fusion protein that avidly binds to the siRNA backbone to mask the negative charge and uses the PTD for macromolecular cellular delivery.

Efficient siRNA delivery into primary cells by a peptide transduction domain-dsRNA binding domain fusion protein.

RNA interference (RNAi) induced by short interfering RNA (siRNA) allows for discovery research and large-scale screening; however, owing to their size and anionic charge, siRNAs do not readily enter cells. Current approaches do not deliver siRNAs into a high percentage of primary cells without cytotoxicity. Here we report an efficient siRNA delivery approach that uses a peptide transduction domain-double-stranded RNA-binding domain (PTD-DRBD) fusion protein. DRBDs bind to siRNAs with high avidity, masking the siRNA's negative charge and allowing PTD-mediated cellular uptake. PTD-DRBD-delivered siRNA induced rapid RNAi in a large percentage of various primary and transformed cells, including T cells, human umbilical vein endothelial cells and human embryonic stem cells. We observed no cytotoxicity, minimal off-target transcriptional changes and no induction of innate immune responses. Thus, PTD-DRBD-mediated siRNA delivery allows efficient gene silencing in difficult-to-transfect primary cell types.

Adaptable molecular interactions guide phosphorylation of the SR protein ASF/SF2 by SRPK1.

The SR (arginine-serine rich) protein ASF/SF2 (also called human alternative splicing factor), an essential splicing factor, contains two functional modules consisting of tandem RNA recognition motifs (RRMs; RRM1-RRM2) and a C-terminal arginine-serine repeat region (RS domain, a domain rich in arginine-serine repeats). The SR-specific protein kinase (SRPK) 1 phosphorylates the RS domain at multiple serines using a directional (C-terminal-to-N-terminal) and processive mechanism--a process that directs the SR protein to the nucleus and influences protein-protein interactions associated with splicing function. To investigate how SRPK1 accomplishes this feat, the enzyme-substrate complex was analyzed using single-turnover and multiturnover kinetic methods. Deletion studies revealed that while recognition of the RS domain by a docking groove on SRPK1 is sufficient to initiate the processive and directional mechanism, continued processive phosphorylation in the presence of building repulsive charge relies on the fine-tuning of contacts with the RRM1-RRM2 module. An electropositive pocket in SRPK1 that stabilizes newly phosphorylated serines enhanced processive phosphorylation of later serines. These data indicate that SRPK1 uses stable, yet highly flexible protein-protein interactions to facilitate both early and late phases of the processive phosphorylation of SR proteins.

Enhancing the cellular uptake of siRNA duplexes following noncovalent packaging with protein transduction domain peptides.

The major limitation in utilizing information rich macromolecules for basic science and therapeutic applications is the inability of these large molecules to readily diffuse across the cellular membrane. While this restriction represents an efficient defense system against cellular penetration of unwanted foreign molecules and thus a crucial component of cell survival, overcoming this cellular characteristic for the intracellular delivery of macromolecules has been the focus of a large number of research groups worldwide. Recently, with the discovery of RNA interference, many of these groups have redirected their attention and have applied previously characterized cell delivery methodologies to synthetic short interfering RNA duplexes (siRNA). Protein transduction domain and cell penetrating peptides have been shown to enhance the delivery of multiple types of macromolecular cargo including peptides, proteins and antisense oligonucleotides and are now being utilized to enhance the cellular uptake of siRNA molecules. The dense cationic charge of these peptides that is critical for interaction with cell membrane components prior to internalization has also been shown to readily package siRNA molecules into stable nanoparticles that are capable of traversing the cell membrane. This review discusses the recent advances in noncovalent packaging of siRNA molecules with cationic peptides and the potential for the resulting complexes to successfully induce RNA interference within both in vitro and in vivo settings.

Exogenous siRNA delivery using peptide transduction domains/cell penetrating peptides.

The cellular membrane constitutes an effective barrier that offers protection for the complex, yet highly ordered, intracellular environment that defines the cell. Passage of molecules across this barrier is highly regulated and highly restricted, with molecular size being the most significant criteria. Over the last 15 years, a class of small cationic peptides has been discovered that can defy the rules of membrane passage and can gain access to the intracellular environment. Importantly, cellular entrance is also permitted for covalently coupled cargo. The cationic nature of these peptides is crucial for their ability to bind and traverse the anionic cellular membrane. Cell penetrating peptides (CPPs) have been used for the delivery of a wide range of macromolecules including peptides, proteins and antisense oligonucleotides. With the recent advancement and understanding of RNA interference (RNAi), CPPs offer an attractive means for the cellular uptake of double-stranded siRNAs to induce a RNAi response. This review focuses on the potential use of CPPs to deliver siRNA into cells and the implications of this technology for both gene function determination and therapeutic potential.

Enhanced targeting and killing of tumor cells expressing the CXC chemokine receptor 4 by transducible anticancer peptides.

Protein transduction domains (PTDs), such as the TAT PTD, have been shown to deliver a wide variety of cargo in cell culture and to treat preclinical models of cancer and cerebral ischemia. The TAT PTD enters cells by a lipid raft-dependent macropinocytosis mechanism that all cells perform. Consequently, PTDs resemble small-molecule therapeutics in their lack of pharmacologic tissue specificity in vivo. However, several human malignancies overexpress specific receptors, including HER2 in breast cancer, GnRH in ovarian carcinomas, and CXC chemokine receptor 4 (CXCR4) in multiple malignancies. To target tumor cells that overexpress the CXCR4 receptor, we linked the CXCR4 DV3 ligand to two transducible anticancer peptides: a p53-activating peptide (DV3-TATp53C') and a cyclin-dependent kinase 2 antagonist peptide (DV3-TAT-RxL). Treatment of tumor cells expressing the CXCR4 receptor with either the DV3-TATp53C' or DV3-TAT-RxL targeted peptides resulted in an enhancement of tumor cell killing compared with treatment with nontargeted parental peptides. In contrast, there was no difference between DV3 targeted peptide and nontargeted, parental peptide treatment of non-CXCR4-expressing tumor cells. These observations show that a multidomain approach can be used to further refine and enhance the tumor selectivity of biologically active, transducible macromolecules for treating cancer.

Treatment of terminal peritoneal carcinomatosis by a transducible p53-activating peptide.

Advanced-stage peritoneal carcinomatosis is resistant to current chemotherapy treatment and, in the case of metastatic ovarian cancer, results in a devastating 15%-20% survival rate. Therapeutics that restore genes inactivated during oncogenesis are predicted to be more potent and specific than current therapies. Experiments with viral vectors have demonstrated the theoretical utility of expressing the p53 tumor suppressor gene in cancer cells. However, clinically useful alternative approaches for introducing p53 activity into cancer cells are clearly needed. It has been hypothesized that direct reactivation of endogenous p53 protein in cancer cells will be therapeutically beneficial, but few tests of this hypothesis have been carried out in vivo. We report that a transducible D-isomer RI-TATp53C' peptide activates the p53 protein in cancer cells, but not normal cells. RI-TATp53C' peptide treatment of preclinical terminal peritoneal carcinomatosis and peritoneal lymphoma models results in significant increases in lifespan (greater than 6-fold) and the generation of disease-free animals. These proof-of-concept observations show that specific activation of endogenous p53 activity by a macromolecular agent is therapeutically effective in preclinical models of terminal human malignancy. Our results suggest that TAT-mediated transduction may be a useful strategy for the therapeutic delivery of large tumor suppressor molecules to malignant cells in vivo.

Anti-cancer protein transduction strategies: reconstitution of p27 tumor suppressor function.

Significant scientific effort focused on understanding the molecular basis of oncogenesis has identified multiple tumor suppressor genes and their corresponding functions. The ultimate goal of this work is to use this knowledge to devise anti-cancer strategies that specifically kill tumor cells in vivo, while leaving normal cells unharmed. Unfortunately, tumor suppressor proteins, while maintaining specificity for their intracellular targets, are often in excess of 20,000 Da and hence, undeliverable in vivo. To address the delivery problem, we previously further developed a protein transduction strategy that allows for the rapid delivery of large, biologically active proteins in excess of 100,000 Da into approximately 100% of cells in culture and most, if not all, cells/tissues in mouse models. The strategy involves the generation of an N-terminal fusion protein that contains the TAT protein transduction domain. Here the ability to manipulate tumor biology in several mouse tumor models in vivo is demonstrated by using protein transduction to delivery the p27(Kip) tumor suppressor protein. These observations serve as a starting point to further develop the delivery of peptide and proteins to specifically treat malignancies in vivo.