Gene-based Drugs Research Articles

Emergence of Small Interfering RNA-Based Gene Drugs for Various Diseases.

Small molecule, peptide, and protein-based drugs have been developed over decades to treat various diseases. The importance of gene therapy as an alternative to traditional drugs has increased after the discovery of gene-based drugs such as Gendicine for cancer and Neovasculgen for peripheral artery disease. Since then, the pharma sector is focusing on developing gene-based drugs for various diseases. After the discovery of the RNA interference (RNAi) mechanism, the development of siRNA-based gene therapy has been accelerated immensely. siRNA-based treatment for hereditary transthyretin-mediated amyloidosis (hATTR) using Onpattro and acute hepatic porphyria (AHP) by Givlaari and three more FDA-approved siRNA drugs has set up a milestone and further improved the confidence for the development of gene therapeutics for a spectrum of diseases. siRNA-based gene drugs have more advantages over other gene therapies and are under study to treat different types of diseases such as viral infections, cardiovascular diseases, cancer, and many more. However, there are a few bottlenecks to realizing the full potential of siRNA-based gene therapy. They include chemical instability, nontargeted biodistribution, undesirable innate immune responses, and off-target effects. This review provides a comprehensive view of siRNA-based gene drugs: challenges associated with siRNA delivery, their potential, and future prospects.

Advances in Nanoparticle Drug Delivery Systems for Anti-Hepatitis B Virus Therapy: A Narrative Review.

Chronic hepatitis B (CHB) is an infectious viral disease that is prevalent worldwide. Traditional nucleoside analogues, as well as the novel drug targets against hepatitis B virus (HBV), are associated with certain critical factors that influence the curative effect, such as biological stability and safety, effective drug delivery, and controlled release. Nanoparticle drug delivery systems have significant advantages and have provided a basis for the development of anti-HBV strategies. In this review, we aim to review the advances in nanoparticle drug delivery systems for anti-hepatitis B virus therapy by summarizing the relevant literature. First, we focus on the characteristics of nanoparticle drug delivery systems for anti-HBV therapy. Second, we discuss the nanoparticle delivery systems for anti-HBV nucleoside drugs, gene-based drugs, and vaccines. Lastly, we provide an overview of the prospects for nanoparticle-based anti-HBV agents.

Gene capsules as functional pharmaceutical formulations for delivery of DNA as an active substance.

Capsules are solid drug forms that are well accepted by patients. Capsules usually consist of a medicinal substance with therapeutic benefits plus excipients. From a pharmaceutical point of view, DNA coding sequences can be treated as active substances. To develop and assess gelatin capsules containing DNA based on the methods included in the Polish Pharmacopoeia XI, as well as non-pharmacopeial methods. The mass uniformity of the obtained capsules was estimated. The stability of the DNA was verified using polymerase chain reaction (PCR) method. A study of the disintegration time of the gene capsules containing DNA mixed with lactose, and placebo capsules with lactose only was also performed. A dissolution test under conditions similar to the nature and specificity of the active substance (DNA) was performed. The transduction activity of the obtained capsules was assessed in cell culture conditions. The DNA and lactose-based gene capsules were obtained. The capsules were characterized by mass uniformity and stability over time. Efficient transduction of B16-F10 cancer cells with the gene product from the capsule was observed using fluorescence microscopy. Groups of green fluorescent protein-positive cells were observed in the microscope field of view. The findings showed that it is possible to obtain a solid pharmaceutical form of the drug, i.e., a capsule containing DNA as an active substance. The gene capsules enabled the introduction of DNA into cells. This method may have valuable implications for increasing the availability of gene-based drugs for patients.

The impact of fusion genes on cancer stem cells and drug resistance.

With ever increasing evidences on the role of fusion genes as the oncogenic protagonists in myriad cancers, it's time to explore if fusion genes can be the next generational drug targets in meeting the current demands of higher drug efficacy. Eliminating cancer stem cells (CSC) has become the current focus; however, we have reached a standstill in drug development owing to the lack of effective strategies to eradicate CSC. We believe that fusion genes could be the novel targets to overcome this limitation. The intriguing feature of fusion genes is that it dominantly impacts every aspect of CSC including self-renewal, differentiation, lineage commitment, tumorigenicity and stemness. Given the clinical success of fusion gene-based drugs in hematological cancers, our attempt to target fusion genes in eradicating CSC can be rewarding. As fusion genes are expressed explicitly in cancer cells, eradicating CSC by targeting fusion genes provides yet an another advantage of negligible patient side effects since normal cells remain unaffected by the drug. We hereby delineate the latest evidences on how fusion genes regulate CSC and drug resistance.

Multi-Target Inhibition of Cancer Cell Growth by SiRNA Cocktails and 5-Fluorouracil Using Effective Piperidine-Terminated Phosphorus Dendrimers

Currently, RNAi based approaches for cancer treatment involving short double stranded RNA molecules (siRNA) are under vigorous scrutinization. Due to numerous biological obstacles, siRNA delivery into target cells requires protective escort. On the other hand, combining of siRNA-mediated gene silencing and action of conventional chemotherapeutics can propose additional enhancement of anticancer activity. In the present study, we investigated a siRNA cocktail able to downregulate anti-apoptotic genes (BCL-xL, BCL-2, MCL-1) and the chemotherapeutic agent 5-fluorouracil (5-FU) to evaluate multi-target cytotoxic effect on human cervical carcinoma cells (HeLa cell line). Novel phosphorus containing dendrimers of 3rd and 4th generations (namely AE2G3 and AE2G4) with voluminous piperidine terminal cationic groups were designed and tested as siRNA carriers. Dendrimers of both generations showed remarkable ability to bind pro-apoptotic siRNAs and provided 80–100% siRNA uptake by HeLa cells in the serum containing medium, while the widespread transfection agent Lipofectamine showed only ~40% uptake. SiRNA cocktail (in low concentrations 50 and 100 nM) delivered by AE2G3 dendrimer caused almost complete elimination of cancer cells. We have discovered considerable increase of 5-FU cytotoxic effect by addition of AE2G3/siRNA cocktail complexes in low doses. Thus, we demonstrated the effectiveness of combined multi-target siRNA anticancer approach and described new highly effective serum stable nanomaterial vehicle for gene-based drugs.

The Advance of Gene Delivery Vectors

Gene therapy has rapidly emerged as a promising method for the treatment of numerous diseases. However, the intrinsic deficiencies of gene-based drugs, such as enzymatic degradation in the body, poor membrane penetrability and lack of target ability, have limited the development of its therapeutic application. Herein, it is important for efficient gene therapy to develop efficient and safe gene vectors that can deliver the gene-based drugs to targeted cells. This paper reviews the current development in gene delivery vectors, especially summarizes the characteristic and development of viral vectors, non-viral vectors and nanoparticles with tracing ability.

Dendritic Peptide-Based Carriers for Gene Delivery

Gene delivery research has come a long way in recent years but there are still a number of barriers preventing therapeutic gene-based drugs reaching the market. Dendrimers or multi-component carriers may provide a means to overcome the issues of gene degradation and transport to the target site by employing different entities in the same carrier. We will discuss some recent approaches to gene delivery using modified dendrimers. We also discuss our work to deliver oligonucleotides and genes using dendritic peptide-based carriers.

Erbium:YAG laser-mediated oligonucleotide and DNA delivery via the skin: An animal study

Topical delivery of antisense oligonucleotides (ASOs) and DNA is attractive for treatment of skin disorders. However, this delivery method is limited by the low permeability of the stratum corneum (SC). The objective of this study was to enhance and optimize the skin absorption of gene-based drugs by an erbium:yttrium-aluminum-garnet (Er:YAG) laser. The animal model utilized nude mice. In the in vitro permeation study, the Er:YAG laser treatment produced a 3–30-fold increase in ASO permeation which was dependent on the laser fluence and ASO molecular mass used. The fluorescence microscopic images showed a more-significant localization of a 15-mer ASO in the epidermis and hair follicles after laser application as compared with the control. The expressions of reporter genes coding for β-galactosidase and green fluorescent protein (GFP) in skin were assessed by X-gal staining and confocal laser scanning microscopy. The SC ablation effect and photomechanical waves produced by the Er:YAG laser resulted in DNA expression being extensively distributed from the epidermis to the subcutis. The GFP expression in 1.4 J/cm 2-treated skin was 160-fold higher than that in intact skin. This non-invasive, well-controlled technique of using an Er:YAG laser for gene therapy provides an efficient strategy to deliver ASOs and DNA via the skin.

Recent Developments in Delivery of Nucleic Acid-Based Antiviral Agents

Rapid advances in viral genomics, gene function and regulation, as well as in rational drug design, have led to the development of gene-based drugs that can induce protective antiviral immunity, interfere with viral replication, suppress viral gene expression or cleave viral mRNAs. Several such drug candidates have been developed in recent years against various viruses including HIV. Although gene-based agents show promise as anti-viral agents their therapeutic efficacy may be restricted by limited delivery to intracellular sites of viral replication and in vivo nuclease degradation. Enhancement of the efficacy of gene-based drugs by encapsulation within liposomes or insertion within viral vectors has been evaluated. This review will highlight recent developments in delivery systems used to target nucleic acid-based drugs into sites of viral replication, therefore avoiding potential drug toxicity in non-viral infected organs. Liposome-encapsulation and insertion of nucleic acid-based drugs within viral vectors can significantly enhance antiviral efficacies. Viral vector-mediated therapy usually results in greater expression of the gene-based drug than with liposome delivery, however significant safety concerns have been raised in regards to viral vector therapies. Research is ongoing to increase drug delivery to the desired target cells while eliminating adverse side effects.

Fusogenic liposome delivers encapsulated nanoparticles for cytosolic controlled gene release

Therapeutic agents based on DNA or RNA oligonucleotides (e.g., antisense DNA oligonucleotide, small interfering RNA) require a regulation of their kinetics in cytoplasm to maintain an optimal concentration during the treatment period. In this respect, delivery of functional nanoparticles containing these drugs into cytoplasm has been thought to have a potential for the cytosolic controlled gene release. In this study, we establish a protocol for the encapsulation of nanoparticles into liposome, which is further fused with ultra violet-inactivated Sendai virus to compose fusogenic liposomes. When nanoparticles were encapsulated in conventional liposomes, endocytosis-mediated uptake of nanoparticles was observed. In contrast, numerous amounts of nanoparticles were delivered into the cytoplasm without any cytotoxicity when the particles were encapsulated in fusogenic liposomes. Additionally, fusogenic liposome showed a high ability to deliver nanoparticles containing DNA oligonucleotides into cytoplasm. These results indicate that this combinatorial nanotechnology using fusogenic liposome and nanoparticle is a valuable system for regulating the intracellular pharmacokinetics of gene-based drugs.

575. In Vivo Evaluation of Supercoiled Plasmids

Top of pageAbstract Supercoiled plasmids are an important component of gene-based drugs. Their applications include gene vaccine and gene therapeutic agents or intermediates in the production of recombinant viruses. These applications require very high quality product with low levels of residual contaminants such as genomic DNA, RNA, host protein and endotoxin. While a variety of methods for the production of supercoiled plasmids for clinical applications have been developed, there is no consensus as to the effect of plasmid quality on biological activity. We have produced two plasmids using four production methodologies based on reversed-phase ion-pairing (PolyFlo) and hydrophobic interaction chromatography, either alone or in tandem processes. The plasmids were analyzed using standard molecular biological techniques as well as more rigorous biophysical techniques. Further, the plasmids were evaluated in vivo in models for gene vaccination and gene therapy. In addition, the gene therapy study included a component to evaluate the degree of plasmid supercoiling and biological activity. Analysis of the plasmids produced by the four methods showed minimal measurable differences using molecular biological methods. Biophysical characterization revealed more significant differences among the preparations. However, there were significant differences in the in vivo response in both models. In the gene vaccination study, preparations using a tandem process of PolyFlo used in two separation modes provided significantly higher antibody responses to an HIV gag antigen. By contrast, PolyFlo as the single chromatographic step resulted in a significant decrease in the rate of tumor growth when compared to two tandem preparations. These data indicate that the process by which supercoiled plasmids are made can influence biological activity and further that the same process might not be ideal for gene vaccine or gene therapy applications. These data emphasize the need for more rigorous methods to evaluate the biological activity of supercoiled plasmids.

561. Analytical Characterization of Supercoiled Plasmids

Supercoiled plasmids are an important component of gene-based drugs. A number of methods for their production for clinical applications have been developed, which require very high quality product with low levels of residual contaminants. In addition, plasmid stability is an essential characteristic when assessing the success of plasmid delivery. However, there is no consensus on the optimal methods to characterize plasmid quality, and further, to determine if these methods are predictive of either product stability or biological activity.

Prophylaxis of acute respiratory virus infections using nucleic acid-based drugs

Acute respiratory virus infections such as SARS and pandemic influenza are highly contagious diseases that cause global crisis, and inflict severe human mortality and morbidity. Vaccines against these viruses are either unavailable or do not provide adequate protection. In the absence of effective vaccines, nucleic acid-based immunomodulators have the potential to offer effective, broad-spectrum protection against these deadly pathogens. Poly ICLC and CpG oligonucleotides are promising gene-based drugs which have been shown in animal studies to protect against acute respiratory virus infections. Poly ICLC is a synthetic double-stranded RNA (dsRNA), and an effective interferon-inducer and natural killer cell activator. When encapsulated in liposomes, poly ICLC offers complete protection (100% survival rate in pretreated group versus 0% survival in control group) against a lethal respiratory challenge of influenza A virus in mice. This antiviral effect has been shown to persist for up to 3 weeks post-drug treatment. Poly ICLC pretreatment also protects mice against a respiratory challenge of western equine encephalitis (WEE) virus, at a level comparable to inactivated WEE vaccine. CpG oligos in liposomes also provided high level of protection against the lethal influenza challenge. Together, these studies suggest nucleic acid-based immunomodulators are promising antiviral agents which can offer effective and non-specific protection against acute respiratory virus infections.

Therapeutic ultrasound: its application in drug delivery.

Ultrasound is best known for its imaging capability in diagnostic medicine. However, there have been considerable efforts recently to develop therapeutic uses for it. The purpose of this review is to summarize some of the recent advances made in the area of therapeutic ultrasound as they relate to drug delivery. In particular, this review will focus on the applications of ultrasound to enhance the delivery and effect of three distinctive therapeutic drug classes: chemotherapeutic, thrombolytic, and gene-based drugs. In addition, ultrasound contrast agents have been recently developed for diagnostic ultrasound. New experimental evidence suggests that these contrast agents can be used as exogenous cavitation nuclei for enhancement of drug and gene delivery. Thus, brief review of this new class of agents and their roles in drug delivery will also be provided. By comparison to diagnostic ultrasound, progress in therapeutic use of ultrasound has been somewhat limited. The recent successes in ultrasound-related drug delivery research positions ultrasound as therapeutic tool for drug delivery in the future.

Assessing the potential of skin electroporation for the delivery of protein- and gene-based drugs

Although transdermal drug delivery has many potential advantages, the permeability of skin to macromolecules is extremely low. However, the application of short, high-voltage pulses to electroporate skin has recently been shown to make it reversibly permeable. A number of studies have demonstrated that electroporation-mediated transdermal delivery of peptides, polysaccharides, oligonucleotides and genes may be possible at clinically relevant rates, leading to the current commercial development of electroporation techniques.

Arsenal of Hope: Revolution in Genetics Creates New Weapons in the War on Cancer

[The following article appeared in The Wall Street Journal, issue of May 6, 1998. Blocking a 'Helper Protein'] The war on cancer, fought for three decades marked by failure and frustration, suddenly is in overdrive. Just a few weeks ago, reports rocked the medical world about two drugs that show promise in preventing breast cancer. This week, Wall Street and Main Street alike went wild over word of a bold experimental drug that wiped out tumors in mice. These approaches are in very early stages of development and, even if all goes well, will require years of human testing before they can move into widespread usage. But they underscore a much bigger story: A quiet revolution in genetics has brought scientists closer than ever before to finding an actual cure for cancer. The drugs that made headlines this week use the promising approach of blocking a tumor's blood vessels. Even farther along in development, however, is a whole new generation of gene-based drugs aimed at a strikingly broad range of cancers. Human testing is already under way for this new arsenal, which looks to be far more powerful and far less toxic than anything tried before.

Synthetic polymers recognize all four base pairs in DNA

Capping 20 years of effort, chemists in Peter B. Dervan's laboratory at California Institute of Technology have completed the design of a chemical recognition code that can distinguish each of the four Watson-Crick base pairs in DNA. They have made small polyamide molecules that can recognize any pre-determined sequence of DNA and bind to it with affinities and specificities similar to those of DNA-binding proteins, the chemists say [ Nature, 391, 468 (1998)]. Such small molecules show major promise as potential gene-based drugs. They might be used, for example, to control the expression of particular genes. The Dervan group showed last year that earlier versions of their polyamides can get into cells and interfere with gene expression. It's an exciting piece of work, says bioorganic chemist Chi-Huey Wong, chemistry professor at Scripps Research Institute, La Jolla, Calif. If these octamers can get into the cell and target the gene, as expected, they will have tremendous potential ...

Stabilization and Regulated Fusion of Liposomes Containing a Cationic Lipid Using Amphipathic Polyethyleneglycol Derivatives

Current fusogenic liposomal delivery systems have limited applicability in vivo due to poor stability in the blood and rapid clearance from the circulation. This is particularly true for liposomes composed of dioleoylphosphatidylethanolamine (DOPE) and cationic lipids, currently being developed for systemic delivery of gene-based drugs. This paper describes a potential strategy to overcome these problems, involving the incorporation of exchangeable amphipathic polyethyleneglycol (PEG) derivatives to transiently stabilize fusogenic liposomes while in the circulation, but where the PEG coating dissipates to reveal fusogenic character at later times after arrival at target sites. It is shown here that large unilamellar vesicles (LUVs) containing Dope and the cationic lipid, N,N-dioleoyl-N, N-dimethylammonium chloride (DODAC) can be stabilized against serum-induced aggregation and fusion by inclusion of at least 2 mol% of PEG coupled to phosphatidylethanolamine (PEG-PE) or ceramide (PEG-Cer). However, low in vitro recovery of fusogenic activity was obtained for the PEG-PE-containing system, presumably due to electrostatic interactions between the negatively charged PEG-PE and the cationic lipid which prevent PEG-PE dissociation from the LUV. Improved recovery of fusogenic activity was achieved for LUVs stabilized by the neutral PEG-Cer derivatives, with shorter chain ceramides exhibiting more rapid recovery rates. Biodistribution studies showed that DOPE/DODAC (85:15, mol/mol) LUVs were rapidly cleared from the circulation, whereas inclusion of 10 mol% PEG-Cer(C20) resulted in significantly prolonged circulation time. Inclusion of shorter ceramide chain lengths resulted in decreased circulation times, consistent with increased exchangeability. These findings demonstrate the feasibility of developing a cationic liposome that is stable in the circulation, but retains its ability to fuse with membranes. This work represents the first step toward the rationale design of fusogenic cationic liposomes for the systemic delivery of gene-based drugs to target tissues, such as tumors.