Focà, M C Liberto, and N Marascio (Italy) 6 Proteomics and Prostate Cancer
Introduction
Organisms experience oxidative stress throughout their lifespan, and the ability of cells to endure this stress relies on the balance between the intensity of the oxidative insult and the effectiveness of their antioxidative defenses Neuronal cell survival is especially critical, as neurons are terminally differentiated and have limited regenerative capacity The brain is particularly susceptible to damage from reactive oxygen species (ROS) due to its high oxygen consumption and relatively weak antioxidant systems Neurons possess various antioxidative mechanisms, including superoxide dismutase (SOD), which converts superoxide radicals into oxygen and hydrogen peroxide, and glutathione reductase/peroxidase (GR/GP), which eliminates hydrogen peroxide Additionally, heme oxygenase (HO) plays a crucial role in degrading heme to produce the antioxidant biliverdin, which is subsequently reduced to bilirubin Notably, depletion of bilirubin through RNA interference significantly increases ROS levels and leads to apoptotic cell death Heme, as a substrate for HO, is found in neurons and is integral to numerous hemoproteins, including cytochrome P450 isoforms CYP3A11 and CYP3A13.
Neuroglobin, a recently discovered monomeric globin with a high affinity for oxygen, is primarily found in the vertebrate brain Stress can lead to the release of free heme from hemoproteins, which is crucial for the antioxidant activity of heme oxygenase (HO) Three catalytically active isoforms of HO—HO-1, HO-2, and HO-3—have been identified, with HO-1 and HO-2 being distinct gene products that differ in primary structure, regulation, and tissue distribution While HO-3 shares approximately 90% homology with HO-2, its function remains unclear Despite their differences, HO-1 and HO-2 play a similar role in catalyzing heme to produce intracellular antioxidants, which are vital for neurons to combat oxidative stress.
Oxidative Stress and Neurodegenerative Diseases
Intracellular accumulation of reactive oxygen species (ROS) plays a crucial role in the pathogenesis of both acute and chronic neurodegenerative diseases, including ischemic stroke and Alzheimer's disease Ischemic stroke, a severe cerebrovascular condition, occurs when blood vessels supplying the brain are blocked, leading to cerebral ischemia and subsequent reperfusion that activates toxic intracellular pathways resulting in cell death The activation of N-methyl-D-aspartate receptors (NMDAr) by glutamate facilitates calcium influx into cells, causing excessive ROS accumulation and mitochondrial dysfunction This disruption in mitochondrial function leads to the production of superoxide and peroxynitrite, contributing to mitochondrial permeability transition and ultimately resulting in apoptotic or necrotic cell death The significant role of ROS in ischemic neurotoxicity is further supported by antioxidant therapies, such as alpha-phenyl-N-tert-butyl nitrone (PBN), which reduce infarct size and mitigate mitochondrial dysfunction, alongside other antioxidants like α-lipoic acid and vitamin E that have been shown to decrease infarct volume in cases of cerebral ischemia.
Alzheimer's disease (AD) typically manifests later in life, beginning with memory deficits and cognitive impairments such as disorientation, poor judgment, and reduced language skills As the disease progresses, patients may exhibit behavioral and personality changes, ultimately leading to motor dysfunction and dementia On average, individuals diagnosed with AD have a life expectancy of 8 to 10 years Pathologically, AD is characterized by brain atrophy and gradual cell loss in the central nervous system, with the formation of extracellular senile plaques being a key finding.
Amyloid β peptides (Aβ 1-42 and Aβ 1-40) and neurofibrillary tangles (NFT), which consist of hyperphosphorylated tau protein, are key features of Alzheimer's disease (AD) that increase in prevalence as the disease advances (Dickson, 1997; Selkoe, 2001; Sisodia et al., 1990; Dowjat et al., 2001; Stoothoff and Johnson, 2005) Oxidative stress, common to AD and other neurodegenerative diseases, contributes significantly to neurological damage (Markesbery and Carney, 1999; Smith et al., 2000) It plays a crucial role in sporadic AD, which constitutes the majority of cases, with the presence of reactive oxygen species (ROS) identified as an early event in the disease's progression (Perry et al., 2000; Nunomura et al., 2001) Additionally, 8-hydroxyguanosine, an oxidized nucleoside derived from RNA, is a notable marker associated with oxidative stress in AD.
Oxidative damage, marked by increased levels of 8-hydroxy-2'-deoxyguanosine (8OHG) and nitrotyrosine, is prevalent in vulnerable neurons of Alzheimer's disease (AD) patients and is considered an early-stage event in neurodegeneration This damage, driven by reactive oxygen species (ROS), contributes to cognitive disturbances and is associated with Mild Cognitive Impairment (MCI), one of the initial stages of AD In affected brain regions, a decline in protein synthesis capabilities coincides with heightened oxidative damage Familial AD is linked to mutations in the amyloid beta protein precursor (AβPP) and presenilin genes, with evidence suggesting that amyloid beta (Aβ) formation enhances ROS production Aβ activates the prooxidative enzyme NADPH-dependent oxidase, resulting in superoxide generation, while hydrogen peroxide (H2O2) is produced during Aβ aggregation Aβ also disrupts metal ion homeostasis and induces lipid peroxidation, leading to the formation of cytotoxic aldehyde 4-hydroxynonenal (4-HNE), which adversely affects calcium homeostasis by impairing membrane calcium pumps and increasing calcium influx through various channels.
Excessive oxidative stress in cells can lead to significant damage, affecting lipids, DNA/RNA, and proteins To combat this, neurons utilize intrinsic antioxidative mechanisms, including enzymatic systems such as superoxide dismutase (SOD) and glutathione reductase/glutathione peroxidase (GR/GP), to eliminate excessive reactive oxygen species (ROS) and prevent related cellular damage.
HO The HO system is an interesting one in which different isoforms of HO come from different genes and cooperates to play an important role in neuronal defense.
Members of the HO Antioxidative System
The heme oxygenase (HO) system consists of three forms: HO-1 (heat shock protein 32), HO-2, and HO-3 HO-1 is a ubiquitous protein whose expression increases significantly in response to heme and cellular stress, making it a key heat shock/stress-response protein It has a molecular weight of 30 kDa and loses 30% of its activity when heated to 60°C for 10 minutes, precipitating in ammonium sulfate at 0–35% saturation In contrast, HO-2 has a molecular weight of 36 kDa, loses 80% of its activity under the same conditions, and precipitates at 35–60% saturation HO-1 and HO-2 are products of different genes, as evidenced by differences in their amino acid compositions, including three cysteine/cystine residues unique to HO-2 Despite sharing 43% amino acid sequence identity, HO-1 is composed of 288 amino acids while HO-2 has 316 HO-1 lacks a signal peptide but has a hydrophobic segment, whereas HO-2 contains two heme-binding sites and a cysteine-proline dipeptide motif Additionally, the genes for HO-1 and HO-2 are located on chromosomes 22q12 and 16p13.3, respectively.
HO-3 is a single-copy gene that produces a transcript of approximately 2.4 kb, encoding a protein of around 33 kDa This transcript is present in various tissues, including the spleen, liver, thymus, prostate, heart, kidney, brain, and testis The amino acid structure of HO-3 is distinct from HO-1 (HSP32) and HO-2, yet it shares a close relationship with HO-2, with about 90% similarity When expressed and purified from Escherichia coli, HO-3 does not cross-react with antibodies against rat HO-1 or HO-2, exhibits poor heme catalytic activity, and has specific hemoprotein spectral characteristics The predicted protein contains two heme regulatory motifs that may play a role in heme binding However, some research indicates that HO-3 may actually be a processed pseudogene derived from HO-2 transcripts.
Distribution of Members of the HO Antioxidative System
Isozymes HO-1 and HO-2 exhibit notable differences in their tissue distribution Under normal conditions, HO-1 is present throughout the brain at low levels and is localized in specific neuronal populations However, in response to stress, HO-1 transcript and protein levels significantly increase, particularly in nonneuronal cells In contrast, HO-2 is exclusively found in neurons within the central nervous system Additionally, research has shown that both HO-1 and HO-2 transcripts are present in neurons and astrocytes, while the HO-3 transcript is uniquely expressed in astrocytes located in the hippocampus, cerebellum, and cortex.
Function of the HO Antioxidative System
Heme oxygenases HO-1 and HO-2 convert heme into biologically active molecules, including iron, biliverdin, and carbon monoxide These products have significant biological effects; for instance, carbon monoxide acts as a potent vasodilator, crucial for regulating vascular tone, particularly in the liver and under stress conditions linked to HO-1 induction Additionally, the release of 'free' iron can heighten oxidative stress and influence the expression of various mRNAs, such as DCT-1, ferritin, and transferrin receptor, by altering the conformation of iron regulatory protein-1 (IRP-1) and its interaction with iron regulatory elements (IREs).
Biliverdin and its derivative bilirubin are powerful antioxidants produced in most mammals, with bilirubin exhibiting neuroprotective properties at nanomolar concentrations and safeguarding cells against excessive hydrogen peroxide (H2O2) Research indicates that HO-2 plays a more crucial role in antioxidation compared to HO-1, as evidenced by lower levels of oxidatively modified proteins in HO-2 cells in response to H2O2 toxicity, despite similar responses to t-BuOOH toxicity Subcellular analysis reveals that HO-2 and NADPH-cytochrome P450 reductase, both vital for heme degradation, are colocalized in the microsome, while HO-1 is only partially present Furthermore, HO-2 transfected cells demonstrate greater resistance to H2O2 than those transfected with HO-1 Notably, under normal conditions, HO-2 is distinctly detectable in the rat brain, unlike HO-1.
FIGURE 1.1 The metabolic pathway of heme.
Expression and Activity Regulations of the HO Antioxidative
The 5′-untranslated region (UTR) of the HO-1 gene contains several regulatory elements, including AP-1, MRE, Myc/Max, ARE, and Sp1 binding sites, which respond to various stressors such as cytokines, hormones, and heavy metals HO-1 expression is notably induced by oxidative stress stimuli, including sodium arsenite, with specific pathways like ERK and p38 being implicated in this process Research indicates that JNK inhibitors can decrease HO-1 mRNA expression in primary rat hepatocytes, while overexpression of certain kinases can enhance HO-1 levels The CRE/AP-1 element is a key site for c-Jun binding, mediating HO-1 induction through the JNK pathway, while p38 isoforms influence HO-1 expression via the E-box Oxidative stress significantly impacts HO-1 levels, with variations depending on cell type and stress intensity; for instance, hydrogen peroxide can dramatically increase HO-1 in astrocytes but not in neurons at lower concentrations Conversely, HO-1 can be down-regulated in specific conditions, such as thermal stress or hypoxia in various human cell lines.
HO-2, primarily found in the brain and testes, is often considered a constitutive protein; however, research indicates that its activity and expression can vary Historically, adrenal glucocorticoids were the only known regulators of HO-2, with corticosterone treatment shown to increase HO-2 mRNA levels Recent studies have demonstrated that HO-2's catalytic activity can be enhanced through phosphorylation by protein kinase C or phorbol esters, leading to increased bilirubin production under oxidative stress Additionally, calmodulin has been identified as a potential regulator of HO-2, binding with high affinity in a calcium-dependent manner and resulting in a threefold increase in its catalytic activity.
Mutations at the binding site hinder calmodulin binding and calcium-dependent enzyme stimulation both in vitro and in cells Calcium-mobilizing agents like ionomycin and glutamate enhance endogenous HO-2 activity in primary cortical cultures (Boehning et al., 2004) Following spinal cord injury (SCI), HO-2 mRNA levels significantly increase proximal to the injury site, peaking at 16 hours post-injury, while HO-1 mRNA levels rise distal to the injury at both time points (Panahian and Maines, 2001) The protein profiles for HO-1 and HO-2 mirror the mRNA distribution patterns In studies comparing HO-2 mRNA levels in cognitively unimpaired and impaired adult and aged rats, both young and aged cognitively impaired rats exhibited elevated expressions in the hippocampus compared to their unimpaired counterparts, with no significant differences observed in the cortices across all groups (Law et al., 2000) However, under specific conditions, HO-2 expression can also be down-regulated, as indicated by real-time PCR showing low levels.
Research indicates that HO-1 and HO-2 mRNA are present in the placenta and deciduas of early gestation CBA/J mice subjected to stress or interleukin 12 exposure (Zenclussen et al., 2002), with HO-2 expression also being suppressed in human pathologic pregnancies (Zenclussen et al., 2003) Studies show that HO-2 protein levels decrease with aging in rat penile tissue (Hu and Han, 2006) and that a 48-hour hypoxic insult reduces HO-2 mRNA and protein expression in human cell lines by shortening the mRNA half-life from 12 to 6 hours (Zhang et al., 2006) Additionally, chronic restraint stress leads to decreased HO-2 protein levels in hippocampal neurons (Chen et al., 2005) Recent findings reveal that HO-2 protein, but not mRNA, is dose-dependently reduced with increased H2O2 concentration under low antioxidant conditions Coordination between HO-1 and HO-2 gene expressions has been reported, where HO-2 may down-regulate HO-1 expression (Ding et al., 2006) Down-regulating HO-2 via siRNA induces HO-1 expression at both mRNA and protein levels by activating its gene promoter, while HO-1 knockdown does not significantly affect HO-2 expression Furthermore, HO-2 is identified as a potent heme metabolic enzyme, as its knockdown leads to heme accumulation in the presence of exogenous hemin, despite the up-regulation of HO-1 HO-3 exhibits very low activity, likely serving a role in heme binding (Mc Coubrey et al., 1997; Kietzmann et al., 2003).
HOs in Neurodegenerative Diseases
Both HO-1 and HO-2 expression decline with age, particularly in critical brain areas like the hippocampus and substantia nigra, which play vital roles in learning and memory Research indicates that older animals exhibit reduced stress responses to hypoxic and hyperthermia conditions, correlating with decreased levels of HO-2 expression.
Heme oxygenase-2 (HO-2) is a vital neuroprotective factor in acute neurodegeneration, particularly in ischemic conditions Deletion of HO-2 (HO-2 −/−) significantly increases neurotoxicity in brain cultures and leads to greater neural damage following transient cerebral ischemia, while HO-1 deletion does not significantly affect stroke damage Additionally, cerebral ischemia can cause reactive oxygen species (ROS) accumulation and apoptosis in cerebral vascular endothelial cells, with HO-2 −/− cells displaying higher basal apoptosis and reduced resistance to serum withdrawal compared to wild-type cells This underscores HO-2's role as an essential endogenous antioxidant in both neurons and endothelial cells Recent studies have also highlighted HO-2's critical neuroprotective functions against collagenase-induced intracerebral hemorrhage and its involvement in mitigating neurodegeneration from traumatic brain injury.
2003) HO-2 activity from injured HO-2 knockout mice was significantly less than that of HO-2 wild types, despite the induction of HO-1 expression after the traumatic brain injury.
In Alzheimer's disease (AD), amyloid-beta (Aβ) is produced from amyloid precursor protein (AβPP), which can interact with heme oxygenase-1 (HO-1) or heme oxygenase-2 (HO-2), leading to a reduction in HO bioactivity (Doré, 2002) Mutations in AβPP associated with familial AD significantly enhance the inhibition of HO activity compared to wild-type AβPP Research on cortical neurons from transgenic mice expressing the Swedish mutant AβPP has demonstrated a notable decrease in bilirubin levels, confirming that mutant AβPP effectively inhibits HO activity.
HO activity in vivo Furthermore, oxidative neurotoxicity is markedly greater in cerebral cortical cultures from AβPP Swedish mutant transgenic mice than wild-type culture (Takahashi et al., 2000).
The HO Antioxidative System as a Drug Target
Numerous antioxidants, including vitamins, coenzyme Q10, and melatonin, have been explored for their potential in treating neurodegeneration (Gilgun-Sherki et al., 2002) Recent research has shifted focus towards inducing HO-1 expression to combat oxidative stress-induced damage, as HO-1 is an inducible enzyme Dietary antioxidants such as α-lipoic acid, cafestol, and curcumin, found in foods like broccoli, coffee, and turmeric, can enhance HO-1 expression (Ogborne et al., 2004) Additionally, pharmacological inducers of HO-1, including simvastatin, aspirin, and sulforaphane, have been identified as potential therapeutic agents (Li et al., 2007).
Maintaining normal intracellular levels of HO-2 is crucial for cellular defense against oxidative stress, which helps protect against ROS-induced neurodegeneration Recent research has shown that the neuroprotective compound PAN-811 effectively prevents oxidative stress-induced neurotoxicity while ensuring that HO-2 protein levels remain at those found in healthy, non-insulted neurons.
Conclusion and Future Perspectives
HO-1 and HO-2, derived from distinct genes, play crucial roles in catalyzing heme to produce antioxidants biliverdin and bilirubin, thereby protecting against oxidative stress-induced neurotoxicity While HO-1 primarily responds to environmental stress, HO-2 is essential for maintaining heme and ROS homeostasis, with HO-2 also influencing HO-1 gene expression Their significant function in reducing excessive intracellular ROS is highlighted by the potent activity of bilirubin, making these heme oxygenases vital targets for neuroprotective drug development Strategies for treatment include inducing HO-1 expression and preserving HO-2 protein levels However, due to the complex mechanisms of neurodegenerative diseases, solely blocking oxidative stress pathways may be inadequate Therefore, co-administering an antioxidative drug with other neuroprotectants, such as NMDA receptor antagonists, could enhance treatment efficacy for neurodegenerative conditions.
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SEARCHING FOR GENE-GENE AND GENE ENVIRONMENT INTERACTIONS
ALISON A MOTSINGER and DAVID M REIF
2.1 Introduction 202.2 Preliminary Analyses 232.3 Traditional Statistical Approaches 262.4 Novel Approaches to Detect Epistasis 322.5 Examples of Epistasis Found in Humans 482.6 Developing an Analysis Plan 49Keywords 50References 51
The search for susceptibility loci in common complex diseases remains a significant challenge in human genetics, often yielding less success compared to simple Mendelian disorders This difficulty arises from various complicating factors, including a higher number of contributing loci, incomplete penetrance, and environmental influences Additionally, gene-gene and gene-environment interactions, known as epistasis, are increasingly recognized as crucial in understanding the etiology of these diseases Epistasis occurs when the effect of one gene is modified by other genetic or environmental factors, complicating the identification of disease-risk variants As interactions between multiple genetic and environmental variables increase, traditional statistical methods become less effective due to the "curse of dimensionality," which leads to sparse data in many contingency table cells Consequently, conventional hierarchical model-building approaches often struggle to adequately address the complexity of epistasis, resulting in a higher likelihood of false negatives and reduced statistical power.
2004) These challenges are magnified by relatively small sample sizes The time and expense involved in sample collection can make effective studies cost prohibitive with traditional analytical methods.
Introduction
RNA interference (RNAi), first discovered by Andrew Fire and Craig Mello in Caenorhabditis elegans, earned them the Nobel Prize in Physiology or Medicine in 2006 This mechanism serves as a defense strategy in plants against viruses and uncontrolled transposon activity Research has shown that double-stranded RNAs (dsRNAs) can effectively knock down specific homologous mRNAs Furthermore, small interfering RNAs (siRNAs), which are 21 to 25 nucleotides long, play a crucial role in the RNAi process The occurrence of RNAi has also been documented in other organisms, including flies.
2002), and vertebrates (Kennerdell et al., 2000; Li et al., 2000) In this chapter, we discuss the RNAi phenomenon in the past, present, and future in therapeutics.
siRNA in Experimental Therapeutics
siRNAs can be exploited in therapeutics to specifically silence genes implicated in transcription and translation of proteins (Pushparaj et al.,
Research has highlighted the potential of RNA interference (RNAi) and small interfering RNAs (siRNAs) in therapeutic applications, particularly for targeting genes involved in disease pathways Numerous studies have reviewed the effectiveness of RNAi in basic research and experimental therapeutics As a result, several Investigational New Drug (IND) applications have been submitted to the US Food and Drug Administration (FDA) seeking approval for clinical trials utilizing siRNAs.
RNA interference (RNAi) therapeutics utilize small interfering RNAs (siRNAs), including synthetic double-stranded RNAs (dsRNAs) and short hairpin RNAs (shRNAs), to specifically target and knock down disease-causing genes These siRNAs bind to and degrade the corresponding mRNA before it can produce harmful proteins associated with illnesses In contrast, microRNAs (miRNAs) are naturally synthesized within cells to regulate essential cellular and molecular processes.
Recent reviews highlight that numerous siRNAs are undergoing clinical trials for diverse conditions, including cancer, ocular diseases, antiviral treatments, kidney disorders, and LDL cholesterol reduction Additionally, research indicates that siRNAs have potential applications across various fields such as infection, immunity, inflammation, neurobiology, cancer biology, and oral biology.
RNA interference (RNAi) has been widely researched in cancer biology and medicine, highlighting its potential in targeting apoptotic genes such as Bcl-2, p53, and Bcr-Abl, which is associated with chronic myelogenous leukemia Additionally, RNAi can silence carcinogenic k-ras transcripts linked to tumors in the colon and pancreas, making it a promising approach for cancer therapy A combinatorial strategy utilizing multiple siRNAs against VEGF-A, VEGFR1, and VEGFR2 has shown enhanced effectiveness in controlling angiogenesis compared to single siRNAs Furthermore, various siRNA targets have been identified to combat vascular diseases and tumor development, underscoring the significance of RNAi in therapeutic applications.
Recent reviews have highlighted the potential of experimental RNA interference (RNAi) in treating neurological diseases, particularly those linked to polyglutamine toxicity, such as Huntington’s disease, where siRNAs have shown effective control (Ramachandran et al., 2014; Deng et al., 2014; Xia et al., 2002; Wood et al., 2003; Davidson and Paulson, 2004) Additionally, RNAi targeting β-secretase (BACE1) has been found to significantly decrease the secretion of β-amyloid peptides Ab140 and Ab142 in vitro, leading to reduced cell death Given that BACE1 is upregulated in Alzheimer’s disease (AD) patients, knockout mice lacking BACE1 demonstrated protection against AD-like symptoms (Kao et al., 2003) Moreover, RNAi targeting of Huntingtin genes in mice has been effective in diminishing the progression of Huntington’s disease (Pushparaj et al., 2008; Yu et al., 2014).
Studies have precisely proved, using hydrodynamic method of siRNA delivery, that liver is the primary organ for siRNA uptake in mice (Zhang et al., 1999;
RNA interference (RNAi) therapeutics have been utilized in the context of fas-mediated apoptosis for various hepatic diseases Research by Chu et al (2005) indicates that small interfering RNAs (siRNAs) targeting fas can significantly reduce the progression of fulminant hepatitis, effectively silencing fas for up to 10 days (Song et al.).
Research has shown that siRNAs targeting caspase 8 can prevent acute liver failure (ALF) in mice, highlighting the potential of RNA interference (RNAi) therapy for treating liver disorders in the future.
Research indicates that small interfering RNAs (siRNAs) are effective in targeting viral genes, as demonstrated in studies involving poliovirus and HIV, which showed that RNA interference (RNAi) strategies can significantly reduce viral propagation Additionally, RNAi therapeutics have been utilized to study the fas cell death receptor and caspase 8, both of which play roles in apoptotic pathways related to acute liver failure caused by viral infections Notably, a combination of siRNAs aimed at the Severe Acute Respiratory Syndrome (SARS) virus has been shown to decrease viral activity, suggesting potential avenues for anti-SARS management and treatment in the future.
The familial amyotrophic lateral sclerosis (ALS) or Lou Gehrig’s disease can be treated using RNAi strategies in the near future In ALS, the mutation in Cu/
Zn superoxide dismutase (SOD1) is linked to motor neuron death, and RNAi silencing of SOD1 has been shown to slow disease progression and reduce mortality in vivo Various strategies for targeting SOD1 mutations include direct mutation targeting, indirect mutation targeting, and addressing aberrant splicing isoforms, all aimed at specifically silencing the mutant SOD1 in amyotrophic lateral sclerosis (ALS).
siRNA Production and Processing Inside the Cell
Long double-stranded RNAs (dsRNAs) are processed into small interfering RNAs (siRNAs) of 21-25 base pairs by the enzyme Dicer These siRNAs are then unwound by helicase and integrated into the RNA-induced silencing complex (RISC), which plays a crucial role in targeting and degrading complementary mRNAs, thereby selectively inhibiting the expression of specific genes.
RNA interference (RNAi) involves the processing of double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) of 21–25 base pairs by the enzyme Dicer These siRNAs are unwound by a helicase and incorporated into the RNA-induced silencing complex (RISC), where they guide the cleavage of complementary mRNA, thereby inhibiting specific mRNA translation Synthetic siRNAs bypass the Dicer step, entering RISC directly for targeted mRNA degradation In mammalian cells, both microRNAs (miRNAs) and siRNAs operate through the same RISC mechanism, facilitating either degradation or translational silencing based on their complementarity to target mRNAs.
Ex Vivo and In Vivo Delivery of siRNAs
We have conducted a thorough review of both ex vivo and in vivo siRNA delivery strategies, highlighting the effectiveness of viral vectors, particularly lentiviral vectors, in delivering siRNAs into blood and bone marrow cells.
The use of viral vectors in RNAi therapeutics faces significant challenges due to toxicity concerns, highlighted by the death of a cancer patient following adenovirus administration Alternative delivery methods, such as cationic liposomes and electroporation, have been explored for the in vitro and in vivo delivery of siRNAs and shRNAs A critical issue remains the tissue-specific delivery of these molecules, which is essential for effective RNAi therapies Electroporation has shown promise in delivering siRNAs to mouse muscle tissues and could be further refined for targeted delivery to specific organs Additionally, siRNAs can be administered in vivo via the portal vein using Lipiodol, through intranasal routes to reach the lungs, or directly applied to the rat brain, all of which have demonstrated the capability to induce RNAi of target genes.
A groundbreaking study by McCaffrey et al (2002) demonstrated the successful in vivo delivery of siRNAs to mouse liver using the hydrodynamic transfection method This approach has been widely utilized in various studies for delivering dsRNAs to different organs in mice (McCaffrey et al., 2002; Golzio et al., 2005) However, the hydrodynamic transfection method poses challenges for clinical applications of siRNAs in humans (Pushparaj et al., 2008).