Intracellular tracking of protamine/antisense oligonucleotide nanoparticles and their inhibitory effect on HIV-1 transactivation.

Membrane transport of antisense oligonucleotides (ODN) is an inefficient process which requires special carriers for their intracellular delivery. We have developed a delivery system for AS-ODN and their phosphorothioate analogues (AS-PTO) directed against human immunodeficiency virus type 1 (HIV-1) tat mRNA for efficient transfection of HIV-1 target cells. Protamine was used to complex AS-ODN and AS-PTO to form nanoparticles with diameters of about 180 nm and surface charges in the range of -18 to +30 mV. Cellular uptake of these nanoparticles was significantly enhanced compared to naked oligonucleotides. A double labeling technique with fluorescently tagged protamine and AS-ODN was used to follow the intracellular fate of the nanoparticles. Protamine/AS-ODN nanoparticles showed release of the antisense compound leading to specific inhibition of tat mediated HIV-1 transactivation. In contrast, protamine/AS-PTO complexes were stable over 72 h, and failed to release AS-PTO. These results demonstrate that protamine/AS-ODN nanoparticles are useful for future therapeutical application to inhibit viral gene expression.     

Genetic therapy for HIV/AIDS

Despite the tremendous success of highly active antiretroviral treatment (HAART) introduced nearly 8 years ago for the treatment of human immunodeficiency virus (HIV), innovative therapies, including gene transfer approaches, are still required for nearly half of the general patient population. A number of potential gene therapeutic targets for HIV have been identified and include both viral and cellular genes essential for viral replication. The diverse methods used to inhibit viral replication comprise RNA-based strategies such as ribozymes, RNA decoys, antisense messenger RNAs and small interfering RNA (siRNA) molecules. Other potential anti-HIV genes include dominant negative viral proteins, intracellular antibodies, intrakines and suicide genes, all of which have had a modicum of success in vitro. Cellular targets include CD4+ T cells, macrophages and their progenitors. The greatest gene transfer efficiency has been achieved using retroviral or, more recently, lentiviral vectors. A limited number of Phase I clinical trials suggest that the general method is safe. It is proposed that a national network for HIV gene therapy (similar to the AIDS Clinical Trial Groups) may be the best way to determine which approaches should proceed clinically.     

In vitro and in vivo intracellular liposomal delivery of antisense oligonucleotides and anticancer drug.

The specific aims of this investigation were (1) to show that conventional and PEGylated liposomes can penetrate cancer cells in vitro and in vivo; (2) to demonstrate that liposomes can be successfully used both for cytoplasmic and nuclear delivery of therapeutics, including anticancer drugs and antisense oligonucleotides; (3) to examine the specific activity of anticancer drugs and nucleotides delivered inside tumor cells by PEGylated liposomes; and (4) to confirm that simultaneous inhibition of pump and nonpump cellular resistance by liposomal ASO can substantially enhance the antitumor activity of traditional well established anticancer drugs in mice bearing xenografts of human multidrug resistant ovarian carcinoma. Experimental results show that PEGylated liposomes are capable of penetrating directly into tumor cells after systemic administration in vivo and do successfully provide cytoplasmic and nuclear delivery of encapsulated anticancer drug (doxorubicin, DOX) and antisense oligonucleotides (ASO). Encapsulation of DOX and ASO into liposomes substantially increased their specific activity. Simultaneous suppression of pump and nonpump resistance dramatically enhanced the ability of DOX for inducing apoptosis leading to higher in vitro cytotoxicity and in vivo antitumor activity.     

Developments in liposomal drug delivery systems

Liposomes are the leading drug delivery systems for the systemic (iv.) administration of drugs. There are now liposomal formulations of conventional drugs that have received clinical approval and many others in clinical trials that bring benefits of reduced toxicity and enhanced efficacy for the treatment of cancer and other life-threatening diseases. The mechanisms giving rise to the therapeutic advantages of liposomes, such as the ability of long-circulating liposomes to preferentially accumulate at disease sites including tumours, sites of infection and sites of inflammation are increasingly well understood. Further, liposome-based formulations of genetic drugs such as antisense oligonucleotides and plasmids for gene therapy that have clear potential for systemic utility are increasingly available. This paper reviews the liposomal drug delivery field, summarises the success of liposomes for the delivery of small molecules and indicates how this success is being built on to design effective carriers for genetic drugs.     

Gene therapy approaches in chronic viral hepatitis

The clinical and socioeconomic background of chronic viral hepatitis is favourable to new therapeutic approaches based on gene biochemistry. As with all gene therapy, the treatment of chronic viral hepatitis using this approach would make use of a therapeutic gene and a delivery system adapted to the pharmaceutical objectives of targeting, gene expression and kinetics. The various vectors under review are not yet sufficiently optimized for selective targeting of infected hepatocytes. Moreover, four therapeutic gene processes are currently under development: antisense oligonucleotides, ribozymes, dominant negative mutants and DNA vaccines. These developments are obviously limited by the lack of experimental or animal models representative of the pathophysiology of chronic viral hepatitis. The gene therapy for chronic viral hepatitis is nearly ready for clinical evaluation but must be weighed against the continuous progress of pharmaceutical treatments.     

Oligonucleotide-based therapeutic options against hepatitis C virus infection

The hepatitis C virus (HCV) is the cause of a silent pandemic that, due to the chronic nature of the disease and the absence of curative therapy, continues to claim an ever-increasing number of lives. Current antiviral regimens have proven largely unsatisfactory for patients with HCV drug-resistant genotypes. It is therefore important to explore alternative therapeutic stratagems whose mode of action allows them to bypass viral resistance. Antisense oligonucleotides, ribozymes, small interfering RNAs, aptamers and deoxyribozymes constitute classes of oligonucleotide-based compounds designed to target highly conserved or functionally crucial regions contained within the HCV genome. The therapeutic expectation for such compounds is the elimination of HCV from infected individuals. Progress in oligonucleotide-based HCV antivirals towards clinical application depends on development of nucleotide designs that bolster efficacy while minimizing toxicity, improvement in liver-targeting delivery systems, and refinement of small-animal models for preclinical testing.     

In vitro and in vivo transport and delivery of phosphorothioate oligonucleotides with cationic liposomes

A recent strategy in gene therapy has been using antiviral genes that are delivered to uninfected cells, either as RNA or DNA, to provide intracellular protection from human immunodeficiency virus type-1 (HIV-1) infection. Antisense oligonucleotides that are complementary to specific target genes suppress gene expression. A variety of techniques are available to enhance the cellular uptake and pharmacological effectiveness of antisense oligonucleotides, both in vitro and in vivo. We investigated the intracellular and tissue uptake of an oligonucleotide/cationic lipid complex, using a fluorescently labeled oligonucleotide. The antisense oligonucleotide was designed against the HIV-1 gag gene sequence. A T-cell line (MT-4) and PHA-stimulated peripheral blood mononuclear cells (PBMCs) were both infected with HIV-1(NL432) at an MOI of 0.01. One h later, both cultures were washed and treated with medium containing 1 microM antisense oligonucleotide. After a 3-day interval, the HIV-1 antigen expression was monitored by an indirect immunofluorescence assay. At 3 days post infection, we confirmed that p24 antigen production was inhibited by the antisense oligonucleotide/cationic lipid complex at a 1/10 ratio in the PBMCs, using enzyme-linked immunosorbent assay (ELISA). We also confirmed the intracellular existence of the complex by fluorescent microscopy. We investigated different means of transporting the antisense oligonucleotide/cationic lipid complex to mouse tissues by intravenous, intraperitoneal and subcutaneous injections. We observed that the anti-HIV-1 activity of the antisense oligonucleotide/cationic lipid complex was the result of enhanced cellular uptake, both in vitro and in vivo. Therefore, the antisense oligonucleotide/cationic lipid complex is an excellent system for the transport and delivery of genes to target cells, as it is effective both in vitro and in vivo.     

Directed HIV-1 evolution of protease inhibitor resistance by second-generation short hairpin RNAs

Despite the success of antiretroviral drugs in decreasing AIDS-related mortality, a substantial fraction of HIV-infected patients experience therapy failure due to the emergence of drug-resistant virus variants. For durable inhibition of HIV-1 replication, the emergence of such escape viruses must be controlled. In addition to antiretroviral drugs, RNA interference (RNAi)-based gene therapy can be used to inhibit HIV-1 replication by targeting the viral RNA genome. RNAi is an evolutionary conserved gene silencing mechanism that mediates the sequence-specific breakdown of the targeted mRNA. Here we investigated an alternative strategy combining the activity of a protease inhibitor (PI) with second-generation short hairpin RNAs (shRNAs) designed to specifically block the emergence of PI-resistant HIV-1 variants. We demonstrate that dominant viral escape routes can be effectively blocked by second-generation shRNAs and that virus evolution can be redirected toward less-fit variants. These results are of importance for a deeper understanding of HIV-1 evolution under combined drug and RNAi pressure and may be used to design future therapeutic approaches.     

Antisense strategy in hematological malignancies.

Standard cytotoxic chemotherapy for neoplastic disease is fraught with systemic toxicity. The ratio of the toxic dose to the therapeutic dose is relatively low, which reflects the large number of cellular targets affected by the chemotherapeutic agent as well as its inability to distinguish between normal and malignant cells. The discovery of oncogenes and tumor suppressor genes involved in the process of transformation of normal cells into malignant cells has opened new areas of research in oncology, aimed at discovering drugs that could selectively inhibit their biological effects. This therapeutic modality, called an antisense strategy, has become a powerful tool for selectively reducing the expression of target genes in vitro, and there is increasing interest in the possibility of using the same technology in vivo for therapeutic purposes. In oncohematology, a number of trials have been initiated with antisense oligonucleotides directed against molecular targets, including the bcl-2, c-myc, bcr-abl, c-myb or p53 oncogenes and tumor suppressor genes. The experience gained from these studies will be applicable to the next generation of antisense compounds, which may include oligonucleotides with novel backbones or other structural modifications, as well as for expansion of the use of antisense oligonucleotides in combination approaches for the treatment of hematological malignancies.     

Immunosuppressive drugs as an adjuvant to HIV treatment

Although highly active antiretroviral therapy (HAART) has dramatically changed the epidemiological impact of HIV infection, many problems with currently used antiretroviral therapy have underscored the urgent need for additional therapeutic approaches. Structured treatment interruption trials, which can be considered an immune-based therapy with an autologous virus, have failed to control viral replication in most chronically HIV-1-infected patients. Alternative approaches could be the use of immunosuppressive drugs to enhance the control of viral replication mediated by their immune and antiviral properties. The use of immunosuppressive drugs may reduce the number of activated CD4 cells that support massive virus production and may prevent sequestration of CD4 T cells into lymphoid tissue, which is the place of antigen presentation and productive HIV infection. The strategy of using drugs that interfere with the HIV life-cycle, acting on the target cells of HIV rather than on viral enzymes, offers the advantage of avoiding the development of antiretroviral drug-resistant HIV mutants. However, it is not known if these approaches will clinically benefit long-term infection, by establishing a new immunological set-point that may affect the rate of disease progression. Caution is required when using HAART in combination with cytostatic drugs in HIV-1 infection until their impact and long-term safety have been investigated further in larger clinical trials.     

Inhibition of HIV-1 replication by simian restriction factors, TRIM5alpha and APOBEC3G

Old World monkey TRIM5alpha targets incoming human immunodeficiency virus type 1 (HIV-1) viral capsid, whereas the apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like (APOBEC)3 family hypermutate/degrade viral sequences. Here, we show the potentials of simian TRIM5alpha and APOBEC3G as therapeutic sequences for AIDS gene therapy. Both rhesus and African green monkey (agm) TRIM5alpha efficiently restrict HIV-1 vectors with divergent Gag from different HIV-1 subtypes. Human T cells genetically engineered to express agm-TRIM5alpha block or delay HIV-1 replication. Although agm-APOBEC3G expression alone only transiently suppresses HIV-1 replication, co-expression of agm-APOBEC3G and agm-TRIM5alpha successfully block the virus replication for more than 5 weeks.     

The development and future of oligonucleotide-based therapies for cervical cancer

Cervical cancer is an attractive model in which to test gene-specific therapies, because elimination of the HPV oncogenes E6 and E7 may result in cancer cell senescence. Oligonucleotide-based therapies tested over the years include antisense oligonucleotides, ribozymes and, more recently, small interfering RNA (siRNA)-based treatments. The development and use of these technologies are reviewed. siRNA-based therapies have been touted as potential treatments for cancers, genetic disorders and viral infections and have a number of advantages over antisense and ribozyme technologies. As with the older technologies, in vitro testing of siRNAs against cervical cancer has shown promising results, however, the issues that held up the clinical development of ribozymes and antisense are currently also challenging the siRNA field; these are target selection, specificity and delivery. If these issues can be overcome, a range of new and potent therapies for cervical cancer could become available.