Non-viral siRNA Delivery Research Articles

Nontoxic, Biodegradable Hyperbranched Poly(β-amino ester)s for Efficient siRNA Delivery and Gene Silencing.

RNA interference (RNAi)-mediated gene silencing is a promising therapeutic approach to treat various diseases, but safe and efficient delivery remains a major challenge to its clinical application. Non-viral gene vectors, such as poly(β-amino esters) (pBAEs), have emerged as a potential candidate due to their biodegradability, low toxicity profile, ease of synthesis, and high gene transfection efficiency for both DNA and siRNA delivery. However, achieving significant gene silencing using pBAEs often requires a large amount of polymer carrier (with polymer/siRNA weight ratio >100) or high siRNA dose (>100 nM), which might potentially exacerbate toxicity concerns during delivery. To overcome these barriers, we designed and optimized a series of hyperbranched pBAEs capable of efficiently condensing siRNA and achieving excellent silencing efficiency at a lower polymer/siRNA weight ratio (w/w) and siRNA dose. Through modulation of monomer combinations and branching density, we identified the top-performing hyperbranched pBAEs, named as h(A2B3)-1, which possess good siRNA condensation ability, low cytotoxicity, and high cellular uptake efficiency. Compared with Lipofectamine 2000, h(A2B3)-1 achieved lower cytotoxicity and higher siRNA silencing efficiency in HeLa cells at a polymer/siRNA weight ratio of 30 and 30 nM siRNA dose. Notably, h(A2B3)-1 enhanced the gene uptake in primary neural cells and effectively silenced the target gene in hard-to-transfect primary cortical neurons and oligodendrocyte progenitor cells, with gene knockdown efficiencies of 34.8 and 53.4% respectively. By incorporating a bioreducible disulfide compartment into the polymer backbone, the cytocompatibility of the h(A2B3)-1 was greatly enhanced while maintaining their good transfection efficiency. Together, the low cytotoxicity and high siRNA transfection efficiency of hyperbranched h(A2B3)-1 in this study demonstrated their great potential as a non-viral gene vector for efficient siRNA delivery and RNAi-mediated gene silencing. This provides valuable insight into the future development of safe and efficient non-viral siRNA delivery systems as well as their translation into clinical applications.

Ongoing Clinical Trials of Nonviral siRNA Therapeutics.

Short interfering RNAs (siRNA) are a powerful class of genetic medicines whose clinical translation can be hindered by their suboptimal delivery properties in vivo. Here, we provide a clinically focused overview that summarizes ongoing siRNA clinical trials from the perspective of innovations in nonviral delivery strategies. More specifically, our review begins by highlighting the delivery barriers and physiochemical properties of siRNA that make it challenging to deliver it in vivo. We then provide commentary on specific delivery strategies, including sequence modification, siRNA ligand conjugation, and nanoparticle and exosomal packaging, each of which can be used to control the delivery of siRNA therapies in living systems. Last, we provide a summary table of ongoing siRNA clinical trials which also highlights the indication of use, target, and National Clinical Trial (NCT) number associated with each entry. In writing this review, our work aims to highlight the key challenges and strategies for effective nonviral siRNA delivery in vivo, while simultaneously summarizing information on ongoing clinical trials for siRNA therapy in humans.

Oleyl Conjugated Histidine-Arginine Cell-Penetrating Peptides as Promising Agents for siRNA Delivery.

Recent approvals of siRNA-based products motivated the scientific community to explore siRNA as a treatment option for several intractable ailments, especially cancer. The success of approved siRNA therapy requires a suitable and safer drug delivery agent. Herein, we report a series of oleyl conjugated histidine–arginine peptides as a promising nonviral siRNA delivery tool. The conjugated peptides were found to bind with the siRNA at N/P ratio ≥ 2 and demonstrated complete protection for the siRNA from early enzymatic degradation at N/P ratio ≥ 20. Oleyl-conjugated peptide -siRNA complexes were found to be noncytotoxic in breast cancer cells (MCF-7 and MDA-MB-231) and normal breast epithelial cells (MCF 10A) at N/P ratio of ~40. The oleyl-R3-(HR)4 and oleyl-R4-(HR)4 showed ~80-fold increased cellular uptake in MDA-MB-231 cells at N/P 40. Moreover, the conjugated peptides-siRNA complexes form nanocomplexes (~115 nm in size) and have an appropriate surface charge to interact with the cell membrane and cause cellular internalization. Furthermore, this study provides a proof-of-concept that oleyl-R5-(HR)4 can efficiently silence STAT-3 gene (~80% inhibition) in MDA-MB-231 cells with similar effectiveness to Lipofectamine. Further exploration of this approach holds a great promise in discovering a successful in vivo siRNA delivery agent with a favorable pharmacokinetic profile.

Microfluidic-Based Cationic Cholesterol Lipid siRNA Delivery Nanosystem: Highly Efficient In Vitro Gene Silencing and the Intracellular Behavior.

Safe and efficient delivery of small interfering RNA (siRNA) is essential to gene therapy towards intervention of genetic diseases. Herein, we developed a novel cationic cholesterol lipid derivative (CEL) in which cholesterol hydrophobic skeleton was connected to L-lysine cationic headgroup via a hexanediol linker as the non-viral siRNA delivery carrier. Well-organized CEL/siRNA nanocomplexes (100–200 nm) were prepared by microfluidic-assisted assembly of CEL and siRNA at various N/P ratios. The CEL and CEL/siRNA nanocomplexes have lower cytotoxicity compared with bPEI25k. Delightfully, we disclosed that, in Hela–Luc and H1299–Luc cell lines, the micro-fluidic-based CEL/siRNA nanocomplexes exhibited high siRNA transfection efficiency under both serum-free condition (74–98%) and low-serum circumstances (80–87%), higher than that of lipofectamine 2000. These nanocomplexes also showed high cellular uptake through the caveolae/lipid-raft mediated endocytosis pathway, which may greatly contribute to transfection efficiency. Moreover, the time-dependent (0–12 h) dynamic intracellular imaging demonstrated the efficient delivery to cytoplasm after lysosomal co-localization. The results indicated that the microfluidic-based CEL/siRNA nanosystems possessed good stability, low cytotoxicity, high siRNA delivery efficiency, rapid cellular uptake and caveolae/lipid raft-dependent internalization. Additionally, this study provides a simple approach for preparing and applying a “helper lipid-free” cationic lipid siRNA delivery system as potential nanotherapeutics towards gene silencing treatment of (tumor) diseases.

Non-Viral Delivery of RNA Gene Therapy to the Central Nervous System.

Appropriate gene delivery systems are essential for successful gene therapy in clinical medicine. Lipid-mediated nucleic acid delivery is an alternative to viral vector-mediated gene delivery and has the following advantages. Lipid-mediated delivery of DNA or mRNA is usually more rapid than viral-mediated delivery, offers a larger payload, and has a nearly zero risk of incorporation. Lipid-mediated delivery of DNA or RNA is therefore preferable to viral DNA delivery in those clinical applications that do not require long-term expression for chronic conditions. Delivery of RNA may be preferable to non-viral DNA delivery in some clinical applications, since transit across the nuclear membrane is not necessary, and onset of expression with RNA is therefore even faster than with DNA, although both are faster than most viral vectors. Delivery of RNA to target organ(s) has previously been challenging due to RNA’s rapid degradation in biological systems, but cationic lipids complexed with RNA, as well as lipid nanoparticles (LNPs), have allowed for delivery and expression of the complexed RNA both in vitro and in vivo. This review will focus on the non-viral lipid-mediated delivery of RNAs, including mRNA, siRNA, shRNA, and microRNA, to the central nervous system (CNS), an organ with at least two unique challenges. The CNS contains a large number of slowly dividing or non-dividing cell types and is protected by the blood brain barrier (BBB). In non-dividing cells, RNA-lipid complexes demonstrated increased transfection efficiency relative to DNA transfection. The efficiency, timing of the onset, and duration of expression after transfection may determine which nucleic acid is best for which proposed therapy. Expression can be seen as soon as 1 h after RNA delivery, but duration of expression has been limited to 5–7 h. In contrast, transfection with a DNA lipoplex demonstrates protein expression within 5 h and lasts as long as several weeks after transfection.

Nonviral siRNA delivery systems for pancreatic cancer therapy.

The serious drawbacks of the conventional treatment of pancreatic ductal adenocarcinoma (PDAC) such as nonspecific toxicity and high resistance to chemo and radiation therapy, have prompted the development and application of countless small interfering RNA (siRNA)-based therapeutics. Recent advances in drug delivery systems hold great promise for improving siRNA-based therapeutics and developing a new class of drugs, known as nano-siRNA drugs. However, many fundamental questions, regarding toxicity, immunostimulation, and poor knowledge of nano-bio interactions, need to be addressed before clinical translation. In this review, we provide recent achievements in the design and development of various nonviral delivery vehicles for pancreatic cancer therapy. More importantly, codelivery of conventional anticancer drugs with siRNA as a new revolutionary pancreatic cancer combinational therapy is completely discussed.

Cuboplex-Mediated Nonviral Delivery of Functional siRNA to Chinese Hamster Ovary (CHO) Cells.

Lipid nanoparticles of internal cubic symmetry, termed cuboplexes, are potential nonviral delivery vehicles for gene therapy due to their "topologically active" nature, which may enhance endosomal escape and improve delivery outcomes. In this study, we have used cationic cuboplexes, based on monoolein (MO) doped with a cationic lipid, for the encapsulation and delivery of antisense green fluorescent protein (GFP)-small interfering RNA (siRNA) into Chinese Hamster Ovary (CHO)-GFP cells. Agarose gel electrophoresis has confirmed the successful encapsulation of siRNA within cationic cubosomes, while synchrotron small-angle X-ray scattering (SAXS) demonstrated that the underlying cubic nanostructure of the particles was retained following encapsulation. The cationic cubosomes were shown to be reasonably nontoxic against the CHO-GFP cell line. Fluorescence-activated cell sorting (FACS) provided evidence of the successful transfection to CHO-GFP cells. Knockdown efficiency was strongly linked to the type of cationic lipid used, although all cubosomes had essentially the same internal nanostructure. The gene knockdown efficiency for some cationic cubosomes was shown to be higher than lipofectamine, which is a commercially available liposome-based formulation, while the controlled release of the siRNA from the cubosomes over a 72 h period was observed using confocal microscopy. This combination exemplifies the potential of cationic cuboplexes as a novel, nonviral, controlled-release delivery vector for siRNA.

Characterization of positively charged polyplexes by tunable resistive pulse sensing

With the approval of the first siRNA-based drugs, non-viral siRNA delivery has gained special interest in industry and academia in the last two years. For non-viral delivery, positively charged lipid and polymer formulations play a central role in research and development. However, nanoparticle size characterization, particularly of polydisperse formulations, can be very challenging. Tunable resistive pulse sensing for particle by particle measurements of size, polydispersity, zeta potential and a direct concentration promises better assessment of nanoparticle formulations. However, the current application is not optimized for positively charged particles. A supplier-provided coating solution for difficult-to-measure samples does not allow for successful measurements of positively charged nanoparticles. This article describes a new coating solution based on choline-chloride. Coating is verified by current–voltage (I-V) recordings and ultimately tested on a positively charged nanoparticle formulation comprising of siRNA and PEG-PCL-PEI polymer. This coating allows successful size, polydispersity index (PDI) and concentration measurement by tunable resistive pulse sensing of positively charged PEI-based polyplexes. This article provides the foundation for further characterization of polyplexes as well as other positively charged nanoparticle formulations based on particle by particle measurements.

Abstract 528: Towards Therapeutic Genome Editing in Vascular Endothelium by Novel Nanoparticle Delivery of Crispr Plasmid Dna

Introduction: Therapeutic delivery of CRISPR system components to induce in vivo genome editing in postnatal and adult life has great translational potential. Recent studies employing non-viral delivery of small guide RNA (gRNA) and Cas9 mRNA have achieved efficient genome editing in adult mice. However, as often seen in other RNA therapeutic studies with non-viral delivery of antisense and siRNA, the efficiency is limited to the liver. Hypothesis: Novel nanoparticle can therapeutically deliver the CRISPR system to selectively target cardiovascular endothelium in adult mice. Methods: We developed novel PLGA-based nanoparticles which was for the first time shown to be uptaken efficiently by the vascular endothelium without specific liver accumulation following i.v. administration. Mixture of the nanoparticle:plasmid DNA expressing Cas9 under the control of the human CDH5 promoter (EC-specific) and gRNA by the U6 promoter was administered i.v. to adult mice. Seven to ten days later, various organ tissues were collected for analysis of the efficiency of genomic editing and knockout of protein expression. The phenotype of CRISPR-mediated in vivo knockout of Pik3cg which encodes the G protein-coupled receptor-activated p110gamma isoform of PI3K was compared to Pik3cg null mice in response to sepsis challenge. Results: Therapeutic delivery of nanoparticles loaded with the all-in-one CRISPR plasmid DNA induced highly efficient genome editing in endothelial cells (ECs) of the cardiovascular system including heart, lung, and aorta in adult mice. The Indel rate was as great as 50% in ECs isolated from these vascular beds. Immunostaining and Western blotting demonstrated greater than 70% decrease of protein expression in ECs. Pik3cg -gRNA-induced genome editing diminished p110γPI3K expression in pulmonary vascular ECs, which led to impaired vascular repair and resolution of inflammation after sepsis challenge as seen in Pik3cg -/- mice. Conclusion: We have developed a simple and highly efficient method for in vivo genome editing selectively targeting the vascular endothelium. This strategy will greatly facilitate cardiovascular research and may enable therapeutic genome editing for prevention and treatment of cardiovascular diseases.

Combinatorial Synthesis of a Lipidoid Library by Thiolactone Chemistry: In Vitro Screening and In Vivo Validation for siRNA Delivery.

Transcriptional inhibition by small interfering RNA (siRNA) delivery using synthetic transfection agents eliminates the subsequent risk of introducing mutations in relevant genes, as opposed to viral vectors. However, synthetic vectors with comparable transfection efficiency to that of viral vectors are yet to be developed. Hence, synthesizing new transfection vehicles with low toxicity is important. In this study, a library of lipid-like molecules (lipidoids) was synthesized by thiolactone chemistry. This library facilitated nonviral delivery of siRNA to mammalian cells, inducing sequence-specific knockdown of a target gene. The liposomal nanoparticles complexed with anti-green fluorescent protein (GFP) siRNA were successfully screened for transfection efficiency using a HeLa-GFP cell line. The five best-performing lipidoids identified in the screening were found to exhibit superior GFP-knockdown efficiency compared with commercially available transfection reagents. The efficiency of siRNA delivery by one of these lipidoids with minimal toxicity was further successfully evaluated in vivo using Kdrl:EGFP zebrafish embryos as a model system. Our study would be important as a facile synthetic route of efficient nonviral nucleic acid delivery to live cells and organisms.

A non-viral nano-delivery system targeting epigenetic methyltransferase EZH2 for precise acute myeloid leukemia therapy.

Acute myeloid leukemia (AML), which is common in the elderly population, accounts for poor long-term survival with a high possibility of relapse. The associated lack of currently developed therapeutics is directing the search for new therapeutic targets relating to AML. EZH2 (Enhancer of Zeste Homolog 2) is a histone methyltransferase member of the polycomb-group (PcG) family, and its significant overexpression in AML means it has emerged as a potential epigenetic target. Here, we propose the human serum albumin (HSA) nanoparticle based delivery of small interfering RNA (siRNA), which can target EZH2-expressing genes in AML. EZH2 specific siRNA loaded in a polyethyleneimine (PEI) conjugated HSA nanocarrier can overcome the systemic instability of siRNA and precisely target the AML cell population for increased EZH2 gene silencing. A stable nanosized complex (HSANPs-PEI@EZH2siRNA), achieved via the electrostatic interaction of PEI and EZH2 siRNA, shows increased systemic stability and hemocompatibility, and enhanced EZH2 gene silencing activity in vitro, compared to conventional transfection reagents. HSANPs-PEI@EZH2siRNA-treated AML cells showed downregulated EZH2, which is associated with a reduced level of Bmi-1 protein, and H3K27me3 and H2AK119ub modification. The ubiquitin-mediated proteasomal degradation pathway plays a critical role in the downregulation of associated proteins following HSANPs-PEI@EZH2siRNA exposure to AML cells. c-Myb is the AML-responsive transcription factor that directly binds on the EZH2 promoter and was downregulated in HSANPs-PEI@EZH2siRNA-treated AML cells. The systemic exposure to HSANPs-PEI@EZH2siRNA of AML engrafted immunodeficient nude mice displayed efficient EZH2 gene silencing and a reduced AML cell population in peripheral blood and bone marrow. The present study demonstrates a non-viral siRNA delivery system for epigenetic targeting based superior anti-leukemic therapy.

Cancer-selective nanoparticles for combinatorial siRNA delivery to primary human GBM in vitro and in vivo

Novel treatments for glioblastoma (GBM) are urgently needed, particularly those which can simultaneously target GBM cells’ ability to grow and migrate. Herein, we describe a synthetic, bioreducible, biodegradable polymer that can package and deliver hundreds of siRNA molecules into a single nanoparticle, facilitating combination therapy against multiple GBM-promoting targets. We demonstrate that siRNA delivery with these polymeric nanoparticles is cancer-selective, thereby avoiding potential side effects in healthy cells. We show that we can deliver siRNAs targeting several anti-GBM genes (Robo1, YAP1, NKCC1, EGFR, and survivin) simultaneously and within the same nanoparticles. Robo1 (roundabout homolog 1) siRNA delivery by biodegradable particles was found to trigger GBM cell death, as did non-viral delivery of NKCC1, EGFR, and survivin siRNA. Most importantly, combining several anti-GBM siRNAs into a nanoparticle formulation leads to high GBM cell death, reduces GBM migration in vitro, and reduces tumor burden over time following intratumoral administration. We show that certain genes, like survivin and EGFR, are important for GBM survival, while NKCC1, is more crucial for cancer cell migration. This represents a powerful platform technology with the potential to serve as a multimodal therapeutic for cancer.

Beyond Trophic Factors: Exploiting the Intrinsic Regenerative Properties of Adult Neurons.

Injuries and diseases of the peripheral nervous system (PNS) are common but frequently irreversible. It is often but mistakenly assumed that peripheral neuron regeneration is robust without a need to be improved or supported. However, axonal lesions, especially those involving proximal nerves rarely recover fully and injuries generally are complicated by slow and incomplete regeneration. Strategies to enhance the intrinsic growth properties of reluctant adult neurons offer an alternative approach to consider during regeneration. Since axons rarely regrow without an intimately partnered Schwann cell (SC), approaches to enhance SC plasticity carry along benefits to their axon partners. Direct targeting of molecules that inhibit growth cone plasticity can inform important regenerative strategies. A newer approach, a focus of our laboratory, exploits tumor suppressor molecules that normally dampen unconstrained growth. However several are also prominently expressed in stable adult neurons. During regeneration their ongoing expression “brakes” growth, whereas their inhibition and knockdown may enhance regrowth. Examples have included phosphatase and tensin homolog deleted on chromosome ten (PTEN), a tumor suppressor that inhibits PI3K/pAkt signaling, Rb1, the protein involved in retinoblastoma development, and adenomatous polyposis coli (APC), a tumor suppressor that inhibits β-Catenin transcriptional signaling and its translocation to the nucleus. The identification of several new targets to manipulate the plasticity of regenerating adult peripheral neurons is exciting. How they fit with canonical regeneration strategies and their feasibility require additional work. Newer forms of nonviral siRNA delivery may be approaches for molecular manipulation to improve regeneration.

Strategies, design, and chemistry in siRNA delivery systems.

Emerging therapeutics that utilize RNA interference (RNAi) have the potential to treat broad classes of diseases due to their ability to reversibly silence target genes. In August 2018, the FDA approved the first siRNA therapeutic, called ONPATTRO™ (Patisiran), for the treatment of transthyretin-mediated amyloidosis. This was an important milestone for the field of siRNA delivery that opens the door for additional siRNA drugs. Currently, >20 small interfering RNA (siRNA)-based therapies are in clinical trials for a wide variety of diseases including cancers, genetic disorders, and viral infections. To maximize therapeutic benefits of siRNA-based drugs, a number of chemical strategies have been applied to address issues associated with efficacy, specificity, and safety. This review focuses on the chemical perspectives behind non-viral siRNA delivery systems, including siRNA synthesis, siRNA conjugates, and nanoparticle delivery using nucleotides, lipids, and polymers. Tracing and understanding the chemical development of strategies to make siRNAs into drugs is important to guide development of additional clinical candidates and enable prolonged success of siRNA therapeutics.

A T7 autogene-based hybrid mRNA/DNA system for long-term shRNA expression in cytoplasm without inefficient nuclear entry

The transient silencing effects currently demonstrated by nonviral siRNA delivery systems limit the therapeutic utility of RNAi, but it remains a technical challenge to prolong duration of gene silencing. We have developed a T7 autogene-based hybrid mRNA/DNA system to enable long-term expression of shRNA in cytoplasm in vitro and in vivo. This hybrid mRNA/DNA system consists of T7 polymerase (T7pol) mRNA, pT7/shRNA-encoding DNA fragment and T7 autogene plasmid, and it can generate higher levels of T7pol proteins, compared to pCMV-triggering T7 autogene system, especially without the need of nuclear entry of any gene. A large amount of T7pol proteins produced are used to induce pT7-driven expression of shRNA in cytoplasm, and through cellular processing of RNA hairpins, mature siRNAs are generated for more than 13 days. We here demonstrate that a single liposomal delivery of this hybrid system leads to the long-term silencing effects in vitro and in vivo, in contrast to the conventional siRNA methods relying on the repeated administrations every 2 or 3 days. These sustainable shRNA expression properties in cytoplasm can provide an efficient strategy to address the limitations caused by shRNA-encoding plasmid DNA systems such as low nuclear entry efficiency and short-term silencing effect. The development of long-term shRNA expression system in vivo could scale down administration frequency of RNAi therapeutics in the treatment of chronic diseases, thereby increasing its clinical utility.

Abstract 17091: High Efficient Therapeutic Genome Editing in Cardiovascular Endotheliumby Nanoparticle Delivery of Crispr Plasmid

Introduction: Therapeutic delivery of CRISPR system components to induce in vivo genome editing in postnatal life has great translational potential. Recent studies employing non-viral delivery of small guide RNA (gRNA) and Cas9 mRNA have achieved efficient in vivo genome editing in adult mice. However, as often seen in other RNA therapeutic studies with non-viral delivery of antisense and siRNA, the efficiency is limited to the liver. Hypothesis: Novel nanoparticle can deliver CRISPR system components in vivo to selectively target cardiovascular endothelium in adult mice. Methods: We developed a novel nanoparticles. Mixture of the nanoparticle:plasmid DNA expressing Cas9 under the control of the human CDH5 promoter and gRNA driven by the U6 promoter was administered i.v. to adult mice. Seven to ten days later, various organ tissues were collected for analysis of the efficiency of genomic editing and knockout of protein expression. The phenotype of CRISPR-mediated in vivo knockout of Pik3cg which encodes the G protein-coupled receptor-activated p110gamma isoform of PI3K was compared to Pik3cg null mice in response to sepsis challenge. Results: Therapeutic delivery of nanoparticles loaded with the all-in-one CRISPR plasmid DNA induced highly efficient genome editing in endothelial cells (ECs) of the cardiovascular system including lung, heart, aorta, and peripheral blood vessels in adult mice. The Indel rate was as great as 50% in ECs isolated from these vascular beds. Immunostaining and Western blotting demonstrated greater than 70% decrease of protein expression in cardiovascular endothelial cells. Pik3cg -gRNA-induced genome editing diminished p110γPI3K expression in pulmonary vascular ECs, which led to impaired vascular repair and resolution of inflammation after sepsis challenge as seen in Pik3cg -/- mice. Conclusion: We have developed a simple and highly efficient method for in vivo genome editing selectively in the cardiovascular endothelium. This strategy will greatly facilitate cardiovascular research and may enable therapeutic genome editing for prevention and treatment of cardiovascular diseases.

Current Non-viral siRNA Delivery Systems as a Promising Treatment of Skin Diseases.

Gene therapy is a new approach to discover and treat many diseases. It has attracted considerable attention from researchers in the last decades. The gene therapy through RNA interference has been considered one of the most recent and revolutionary approaches used in individualized therapy. In the last years, we have witnessed the rapid development in the field of the gene silencing and knockdown by topical siRNA. Its application in gene therapy has become an attractive alternative for drug development. This article will address topical delivery of siRNA as a promising treatment for skin disorders. An update on the advances in siRNA-based nanocarriers as a powerful therapeutic strategy for several skin diseases will be discussed giving emphasis on in vitro evaluations. Through the in-depth review of the literature on the use of siRNAs for skin diseases we realize how widespread this use is. We have also realized that nanoparticles as non-viral vectors are increasingly being explored. Skin diseases where the use of siRNA has been explored most are skin cancer (melanoma and nonmelanoma), psoriasis, vitiligo, dermatitis and leprosy. But we also report here other diseases where the use of siRNA has been growing as acne, alopecia areata, cutaneous leishmaniasis, mycoses, herpes, epidermolysis bullosa and oculocutaneous albinism. Also highlighted, the first clinical trial of siRNA for cutaneous diseases, aimed at Pathyounychia Congenita. The treatment of skin diseases based on topical delivery of siRNA, which act by inhibiting the expression of target transcripts, offers many potential therapeutic advantages for suppressing genes into the skin.

Optimal model-based control of non-viral siRNA delivery.

Further quantitative understanding of the biological effects and mechanisms involved in cellular and intracellular delivery of nucleic acid materials is required to produce clinical applications of gene therapy. Several modeling approaches have been used in this field; however, a comprehensive approach that integrates all the key pharmacological issues into a holistic framework that is applicable for in vivo conditions is still lacking. This contribution presents a pharmacokinetic/pharmacodynamic model-based control study of non-viral siRNA delivery describing the dynamics of the delivery process and takes into account the main multi-objective optimization issues such as efficacy and toxicity, as well as the effect of uncertainty in cell doubling time. The methodology developed in this work is used to predict the optimal dosage injection rate and optimal intracellular exposure of siRNAs in order to improve the pharmacological effects before cell division occurs. The present analysis successfully provides quantitative predictions of non-viral siRNA activity paving the path for further experimental work to probe more efficient delivery systems.

In vivo therapeutic efficacy of TNFα silencing by folate-PEG-chitosan-DEAE/siRNA nanoparticles in arthritic mice.

BackgroundTumor necrosis factor-alpha (TNFα), a pro-inflammatory cytokine, has been shown to play a role in the pathophysiology of rheumatoid arthritis. Silencing TNFα expression with small interfering RNA (siRNA) is a promising approach to treatment of the condition.MethodsTowards this end, our team has developed a modified chitosan (CH) nanocarrier, deploying folic acid, diethylethylamine (DEAE) and polyethylene glycol (PEG) (folate-PEG-CH-DEAE15). The gene carrier protects siRNA against nuclease destruction, its ligands facilitate siRNA uptake via cell surface receptors, and it provides improved solubility at neutral pH with transport of its load into target cells. In the present study, nanoparticles were prepared with siRNA-TNFα, DEAE, and folic acid-CH derivative. Nanoparticle size and zeta potential were verified by dynamic light scattering. Their TNFα-knockdown effects were tested in a murine collagen antibody-induced arthritis model. TNFα expression was examined along with measurements of various cartilage and bone turnover markers by performing histology and microcomputed tomography analysis.ResultsWe demonstrated that folate-PEG-CH-DEAE15/siRNA nanoparticles did not alter cell viability, and significantly decreased inflammation, as demonstrated by improved clinical scores and lower TNFα protein concentrations in target tissues. This siRNA nanocarrier also decreased articular cartilage destruction and bone loss.ConclusionThe results indicate that folate-PEG-CH-DEAE15 nanoparticles are a safe and effective platform for nonviral gene delivery of siRNA, and their potential clinical applications warrant further investigation.

Systemic siRNA delivery to tumors by cell-penetrating α-helical polypeptide-based metastable nanoparticles.

Systemic, non-viral siRNA delivery for cancer treatment is mainly achieved via condensation by cationic materials (e.g., lipids and cationic polymers), which nevertheless, suffers from poor serum stability, non-specific tissue interaction, and unsatisfactory membrane activity against efficient in vivo gene knockdown. Here, we report the design of a metastable, cancer-targeting siRNA delivery system based on two functional polymers, PVBLG-8, a cationic, helical cell-penetrating polypeptide, and poly(l-glutamic acid) (PLG), an anionic random-coiled polypeptide. PVBLG-8 with rigid, linear structure showed weak siRNA condensation capability, and PLG with flexible chains was incorporated as a stabilizer which provided sufficient molecular entanglement with PVBLG-8 to encapsulate the siRNA within the polymeric network. The obtained PVBLG-8/siRNA/PLG nanoparticles (PSP NPs) with positive charges were sequentially coated with additional amount of PLG, which reversed the surface charge from positive to negative to yield the metastable PVBLG-8/siRNA/PLG@PLG (PSPP) NPs. The PSPP NPs featured desired serum stability during circulation to enhance tumor accumulation via the enhanced permeability and retention (EPR) effect. Upon acidification in the tumor extracellular microenvironment and intracellular endosomes, the partial protonation of PLG on PSPP NPs surface would lead to dissociation of PLG coating from NPs, exposure of the highly membrane-active PVBLG-8, and surface charge reversal from negative to positive, which subsequently promoted tumor penetration, selective cancer cell internalization, and efficient endolysosomal escape. When siRNA against epidermal growth factor receptor (EGFR) was encapsulated, the PSPP NPs showed excellent tumor penetration capability, tumor cell uptake level, EGFR silencing efficiency, and tumor growth inhibition efficacy in U-87 MG glioblastoma tumor spheroids in vitro and in xenograft tumor-bearing mice in vivo, outperforming the PSP NPs and several commercial reagents such as Lipofectamine 2000 and poly(l-lysine) (PLL). This study therefore demonstrates a facile and unique design approach of metastable and charge reversal NPs, which overcomes multiple biological barriers against systemic siRNA delivery toward anti-cancer treatment.