Stable nucleic acid lipid particles (SNALPs) are microscopic particles approximately 120 nanometers in diameter, smaller than the wavelengths of visible light. They have been used to deliver siRNAs therapeutically to mammals in vivo. In SNALPs, the siRNA is surrounded by a lipid bilayer containing a mixture of cationic and fusogenic lipids, coated with diffusible polyethylene glycol. [1]
RNA interference(RNAi) is a process that occurs naturally within the cytoplasm inhibiting gene expression at specific sequences. Regulation of gene expression through RNAi is possible by introducing small interfering RNAs(siRNAs), which effectively silence expression of a targeted gene. RNAi activates the RNA-induced silencing complex(RISC) containing siRNA, siRNA derived from cleaved dsRNA. The siRNA guides the RISC complex to a specific sequence on the mRNA that is cleaved by RISC and, consequently, silences those genes. [2]
However, without modifications to the RNA backbone or inclusion of inverted bases at either end, siRNA instability in the plasma makes it extremely difficult to apply this technique in vivo. Pattern recognition receptors(PRRs), which can be grouped as endocytic PRRs or signaling PRRs, are expressed in all cells of the innate immune system. Signaling PRRs, in particular, include Toll-like receptors(TLRs) and are involved primarily with identifying pathogen-associated molecular patterns(PAMPs). For example, TLRs can recognize specific regions conserved in various pathogens, recognition stimulating an immune response with potentially devastating effects to the organism. In particular, TLR 3 recognizes both dsRNA characteristic of viral replication and siRNA, which is also double-stranded. [3] In addition to this instability, another limitation of siRNA therapy concerns the inability to target a tissue with any specificity.
SNALPs, though, may provide the stability and specificity required for this mode of RNAi therapy to be effective. Consisting of a lipid bilayer, SNALPs are able to provide stability to siRNAs by protecting them from nucleases within the plasma that would degrade them. In addition, delivery of siRNAs is subject to endosomal trafficking, potentially exposing them to TLR3 and TLR7, and can lead to activation of interferons and proinflammatory cytokines. However, SNALPs allow siRNA uptake into the endosome without activating Toll-like receptors and consequently stimulating an impeding immune response, thus enabling siRNA escape from the endosome. [1]
Downregulation of gene expression via siRNA has been an important research tool in in vitro studies. Susceptibility of siRNAs to nuclease degradation, though, makes use of them in vivo problematic. In 2005, researchers working with hepatitis B virus(HBV) in rodents, determined that certain modifications of the siRNA prevented degradation by nucleases within the plasma and lead to increased gene silencing compared to unmodified siRNA. Modifications to the sense and antisense strands were made differentially. With respect to both sense and antisense strands, 2'-OH was substituted with 2'-fluoro at all pyrimidine positions. In addition, sense strands were modified at all purine positions with deoxyribose, antisense strands modified with 2'-O-methyl at the same positions. The 5' and 3' ends of the sense strand were capped with abasic inverted repeats, while a phosphorothioate linkage was incorporated at the 3' end of the antisense strand. [4]
Although this research demonstrated a potential RNAi therapy using modified siRNA, the 90% reduction in HBV DNA in rodents resulted from a 30 mg/kg dosage with frequent administration. Because this is not a viable dosing regime, this same group looked at the effects of encapsulating the siRNA in a PEGylated lipid bilayer, or SNALP. Specifically, the lipid bilayer facilitates uptake into the cell and subsequent release from the endosome, the PEGylated outer layer providing stability during formulation due to the resulting hydrophilicity of the exterior. According to this 2005 study, researchers obtained 90% reduction in HBV DNA with a 3 mg/kg/day dose of siRNA for three days, a dose substantially lower than the earlier study. In addition, in contrast to unmodified or modified and non-encapsulated siRNA, administration of SNALP-delivered siRNA resulted in no detectable levels of interferons, such as IFN-a, or inflammatory cytokines associated with immunostimulation. Even so, researchers acknowledged that more work was necessary in order to reach a feasible dose and dosing regime. [5]
In 2006, researchers working on silencing of apolipoprotein B(ApoB) in non-human primates achieved 90% silencing with a single dose of 2.5 mg/kg of SNALP-delivered APOB-specific siRNA. ApoB is a protein involved with the assembly and secretion of very-low-density lipoprotein(VLDL) and low-density lipoprotein(LDL), and it is expressed primarily in the liver and jejunum. Both VLDL and LDL are important in cholesterol transport and its metabolism. Not only was this degree of silencing observed very quickly, in about 24 hours post-administration, but the silencing effects maintained for over 22 days after only a single dose. Researchers tested a 1 mg/kg single dose, too, obtaining a 68% silencing of the target gene, indicating dose-dependent silencing. This dose-dependent silencing was evident not only on the degree of silencing but the duration of silencing, expression of the target gene recovering 72 hours post-administration. [6]
Although SNALPs having a 100 nm diameter have been used effectively to target specific genes for silencing, there are a variety of systemic barriers that relate specifically to size. For example, diffusion into solid tumors is impeded by large SNALPs and, similarly, inflamed cells having enhanced permeation and retention make it difficult for large SNALPs to enter. In addition, reticuloendothelial elimination, blood–brain barrier size-selectivity and limitations of capillary fenestrae all necessitate a smaller SNALP in order to effectively deliver target-specific siRNA. In 2012, scientists in Germany developed what they termed "mono-NALPs" using a fairly simple solvent exchange method involving progressive dilution of a 50% isopropanol solution. What results is a very stable delivery system similar to traditional SNALPs, but one having only a diameter of 30 nm. The mono-NALPs developed here, however, are inactive, but can become active carriers by implementing specific targeting and release mechanisms used by similar delivery systems. [7]
We were able to confer complete protection with either a pool of siRNAs encapsulated in SNALPs or individual SNALP siRNAs, depending on their relative potency ... [the most potent siRNA] ... conferred absolute protection, that is 100 percent survival, and also contributed to complete aviremia in the infected guinea pigs. So there was no detectable Ebola virus even though the animals had been inoculated with essentially 30,000 times the lethal infectious dose for the virus.
In May 2010, an application of SNALPs to the Ebola Zaire virus made headlines, as the preparation was able to cure rhesus macaques when administered shortly after their exposure to a lethal dose of the virus, which can be up to 90% lethal to humans in sporadic outbreaks in Africa. The treatment used for rhesus macaques consisted of three siRNAs (staggered duplexes of RNA) targeting three viral genes. The SNALPs (around 81 nm in size here) were formulated by spontaneous vesiculation from a mixture of cholesterol, dipalmitoyl phosphatidylcholine, 3-N-[(ω-methoxy poly(ethylene glycol)2000)carbamoyl]-1,2-dimyrestyloxypropylamine, and cationic 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane. [9]
In addition to the rhesus macaque application, SNALPs have also been proven to protect cavia porcellua from viremia and death when administered shortly after postexposure to ZEBOV. A polymerase (L) gene-specific siRNAs delivery system was imposed upon four genes associated with the viral genomic RNA in the ribonucleoprotein complex found within EBOV particles (three of which match the application above): NP, VP30, VP35, and the L protein. The SNALPs ranged from 71 – 84 nm in size and were composed of synthetic cholesterol, phospholipid DSPC, PEG lipid PEGC-DMA, and cationic lipid DLinDMA at the molar ratio of 48:20:2:30. [10] The results confirm complete protection against viremia and death in guinea pigs when administered a SNALP-siRNA delivery system after diagnosis of the Ebola virus, thus proving this technology to be an effective treatment. Future studies will focus mainly upon evaluating the effects of siRNA ‘cocktails’ on EBOV genes to increase antiviral effects. [10]
In 2010, researchers developed an applicable targeting therapy for hepatocellular carcinoma (HCC) in humans. The identification of CSN5, the fifth subunit of the COP9 signalosome complex found in early HCC, was used as a therapeutic target for siRNA induction. Systemic delivery of modified CSN5siRNA encapsulated in SNALPs significantly inhibited hepatic tumor growth in the Huh7-luc+ orthotopic xenograft model of human liver cancer. SiRNA-mediated CSN5 knockdown was also proven to inhibit cell-cycle progression and increases the rate of apoptosis in HCC cells in vitro. Not only do these results demonstrate the role of CSN5 in liver cancer progression, they also indicate that CSN5 has an essential role in HCC pathogenesis. In conclusion, SNALPs have been proven to significantly reduce hepatocellular carcinoma tumor growth in human Huh7-luc* cells through therapeutic silencing. [11]
In 2009, researchers developed siRNAs capable of targeting both polo-like kinase 1(PLK1) and kinesin spindle protein(KSP). Both proteins are important to the cell-cycle of tumor cells, PLK1 involved with phosphorylation of a variety of proteins and KSP integral to chromosome segregation during mitosis. Specifically, bipolar mitotic spindles are unable to form when KSP is inhibited, leading to arrest of the cell cycle and, eventually, apoptosis. Likewise, inhibition of PLK1 facilitates mitotic arrests and cell apoptosis. According to the study, a 2 mg/kg dose of PLK1-specific siRNA administered for 3 weeks to mice implanted with tumors resulted in increased survival times and obvious reduction of tumors. In fact, the median survival time of treated mice was 51 days as opposed to 32 days for the controls. Further, only 2 of the 6 mice treated had noticeable tumors around the implantation site. Even so, GAPDH, a tumor-derived signal, was present at low levels, indicating significant suppression of tumor growth but not complete elimination. Still, the results suggested minimal toxicity and no significant dysfunction of the bone marrow. Animals treated with KSP-specific siRNA, too, exhibited increased survival times of 28 days compared to 20 days in the controls. [12]
Gene silencing is the regulation of gene expression in a cell to prevent the expression of a certain gene. Gene silencing can occur during either transcription or translation and is often used in research. In particular, methods used to silence genes are being increasingly used to produce therapeutics to combat cancer and other diseases, such as infectious diseases and neurodegenerative disorders.
Gene knockdown is an experimental technique by which the expression of one or more of an organism's genes is reduced. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript.
Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA at first non-coding RNA molecules, typically 20-24 base pairs in length, similar to miRNA, and operating within the RNA interference (RNAi) pathway. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, preventing translation.
Dicer, also known as endoribonuclease Dicer or helicase with RNase motif, is an enzyme that in humans is encoded by the DICER1 gene. Being part of the RNase III family, Dicer cleaves double-stranded RNA (dsRNA) and pre-microRNA (pre-miRNA) into short double-stranded RNA fragments called small interfering RNA and microRNA, respectively. These fragments are approximately 20–25 base pairs long with a two-base overhang on the 3′-end. Dicer facilitates the activation of the RNA-induced silencing complex (RISC), which is essential for RNA interference. RISC has a catalytic component Argonaute, which is an endonuclease capable of degrading messenger RNA (mRNA).
Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred: "transformation" is typically used to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells, including plant cells. In animal cells, transfection is the preferred term as transformation is also used to refer to progression to a cancerous state (carcinogenesis) in these cells. Transduction is often used to describe virus-mediated gene transfer into eukaryotic cells.
Antisense RNA (asRNA), also referred to as antisense transcript, natural antisense transcript (NAT) or antisense oligonucleotide, is a single stranded RNA that is complementary to a protein coding messenger RNA (mRNA) with which it hybridizes, and thereby blocks its translation into protein. asRNAs have been found in both prokaryotes and eukaryotes, and can be classified into short and long non-coding RNAs (ncRNAs). The primary function of asRNA is regulating gene expression. asRNAs may also be produced synthetically and have found wide spread use as research tools for gene knockdown. They may also have therapeutic applications.
The RNA-induced silencing complex, or RISC, is a multiprotein complex, specifically a ribonucleoprotein, which functions in gene silencing via a variety of pathways at the transcriptional and translational levels. Using single-stranded RNA (ssRNA) fragments, such as microRNA (miRNA), or double-stranded small interfering RNA (siRNA), the complex functions as a key tool in gene regulation. The single strand of RNA acts as a template for RISC to recognize complementary messenger RNA (mRNA) transcript. Once found, one of the proteins in RISC, Argonaute, activates and cleaves the mRNA. This process is called RNA interference (RNAi) and it is found in many eukaryotes; it is a key process in defense against viral infections, as it is triggered by the presence of double-stranded RNA (dsRNA).
A short hairpin RNA or small hairpin RNA is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors. shRNA is an advantageous mediator of RNAi in that it has a relatively low rate of degradation and turnover. However, it requires use of an expression vector, which has the potential to cause side effects in medicinal applications.
In molecular biology and genetics, the sense of a nucleic acid molecule, particularly of a strand of DNA or RNA, refers to the nature of the roles of the strand and its complement in specifying a sequence of amino acids. Depending on the context, sense may have slightly different meanings. For example, negative-sense strand of DNA is equivalent to the template strand, whereas the positive-sense strand is the non-template strand whose nucleotide sequence is equivalent to the sequence of the mRNA transcript.
The Argonaute protein family, first discovered for its evolutionarily conserved stem cell function, plays a central role in RNA silencing processes as essential components of the RNA-induced silencing complex (RISC). RISC is responsible for the gene silencing phenomenon known as RNA interference (RNAi). Argonaute proteins bind different classes of small non-coding RNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). Small RNAs guide Argonaute proteins to their specific targets through sequence complementarity, which then leads to mRNA cleavage, translation inhibition, and/or the initiation of mRNA decay.
RNA silencing or RNA interference refers to a family of gene silencing effects by which gene expression is negatively regulated by non-coding RNAs such as microRNAs. RNA silencing may also be defined as sequence-specific regulation of gene expression triggered by double-stranded RNA (dsRNA). RNA silencing mechanisms are highly conserved in most eukaryotes. The most common and well-studied example is RNA interference (RNAi), in which endogenously expressed microRNA (miRNA) or exogenously derived small interfering RNA (siRNA) induces the degradation of complementary messenger RNA. Other classes of small RNA have been identified, including piwi-interacting RNA (piRNA) and its subspecies repeat associated small interfering RNA (rasiRNA).
RNA interference (RNAi) is a biological process in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA, through translational or transcriptional repression. Historically, RNAi was known by other names, including co-suppression, post-transcriptional gene silencing (PTGS), and quelling. The detailed study of each of these seemingly different processes elucidated that the identity of these phenomena were all actually RNAi. Andrew Fire and Craig C. Mello shared the 2006 Nobel Prize in Physiology or Medicine for their work on RNAi in the nematode worm Caenorhabditis elegans, which they published in 1998. Since the discovery of RNAi and its regulatory potentials, it has become evident that RNAi has immense potential in suppression of desired genes. RNAi is now known as precise, efficient, stable and better than antisense therapy for gene suppression. Antisense RNA produced intracellularly by an expression vector may be developed and find utility as novel therapeutic agents.
Gene therapy utilizes the delivery of DNA into cells, which can be accomplished by several methods, summarized below. The two major classes of methods are those that use recombinant viruses and those that use naked DNA or DNA complexes.
A "classifier" is created to categorize cells by identifying specific characteristics of cervical cancer. These characteristics are consistent with HeLa cells, which serve as the target cell line for cell death. Upon identifying these cells, the classifier releases specific proteins within the HeLa cell that trigger apoptosis without killing or endangering neighboring, healthy cells.
RDE-1 is a primary Argonaute protein required for RNA-mediated interference (RNAi) in Caenorhabditis elegans. The rde-1 gene locus was first characterized in C. elegans mutants resistant to RNAi, and is a member of a highly conserved Piwi gene family that includes plant, Drosophila, and vertebrate homologs.
DNA-directed RNA interference (DRNAI) is a gene-silencing technique that utilizes DNA constructs to activate an animal cell's endogenous RNA interference (RNAI) pathways. DNA constructs are designed to express self-complementary double-stranded RNAs, typically short-hairpin RNAs, that once processed bring about silencing of a target gene or genes. Any RNA, including endogenous messenger RNA (mRNAs) or viral RNAs, can be silenced by designing constructs to express double-stranded RNA complementary to the desired mRNA target.
Cancer treatments may vary depending on what type of cancer is being targeted, but one challenge remains in all of them: it is incredibly difficult to target without killing good cells. Cancer drugs and therapies all have very low selective toxicity. However, with the help of nanotechnology and RNA silencing, new and better treatments may be on the horizon for certain forms of cancer.
RNA therapeutics are a new class of medications based on ribonucleic acid (RNA). Research has been working on clinical use since the 1990s, with significant success in cancer therapy in the early 2010s. In 2020 and 2021, mRNA vaccines have been developed globally for use in combating the coronavirus disease. The Pfizer–BioNTech COVID-19 vaccine was the first mRNA vaccine approved by a medicines regulator, followed by the Moderna COVID-19 vaccine, and others.
Intracellular delivery is the process of introducing external materials into living cells. Materials that are delivered into cells include nucleic acids, proteins, peptides, impermeable small molecules, synthetic nanomaterials, organelles, and micron-scale tracers, devices and objects. Such molecules and materials can be used to investigate cellular behavior, engineer cell operations or correct a pathological function.
DCL2 is a gene in plants that codes for the DCL2 protein, a ribonuclease III enzyme involved in processing exogenous double-stranded RNA (dsRNA) into 22 nucleotide small interference RNAs (siRNAs).