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Human Cytomegalovirus (HCMV) infection may result in severe outcomes in immunocompromised individuals such as AIDS patients, transplant recipients, and neonates. To date, no vaccines are available and there are only few drugs for anti-HCMV therapy. Adverse effects and the continuous emergence of drug-resistance strains require the identification of new drug candidates in the near future. Identification and characterization of such compounds and biological factors requires sensitive and reliable detection techniques of HCMV infection, gene expression and spread. In this work, we present and validate a novel concept for multi-reporter herpesviruses, identified through iterative testing of minimally invasive mutations. We integrated up to three fluorescence reporter genes into replication-competent HCMV strains, generating reporter HCMVs that allow the visualization of replication cycle stages of HCMV, namely the immediate early (IE), early (E), and late (L) phase. Fluorescent proteins with clearly distinguishable emission spectra were linked by 2A peptides to essential viral genes, allowing bicistronic expression of the viral and the fluorescent protein without major effects on viral fitness. By using this triple color reporter HCMV, we monitored gene expression dynamics of the IE, E, and L genes by measuring the fluorescent signal of the viral gene-associated fluorophores within infected cell populations and at high temporal resolution. We demonstrate distinct inhibitory profiles of foscarnet, fomivirsen, phosphonoacetic acid, ganciclovir, and letermovir reflecting their mode-of-action. In conclusion, our data argues that this experimental approach allows the identification and characterization of new drug candidates in a single step.

A Brief History of Genetic Therapy: Gene Therapy, Antisense Technology, and Genomics, James W. Hawkins Preclinical Development of Oligonucleotide Therapeutics for Cancer: Regulatory Aspects, Chang-Ho Ahn and Joseph J. DeGeorge Commercial Scale Manufacturing of Oligonucleotides Under Good Manufacturing Practices, Jose E. Gonzalez, Richard G. Einig, Patricia Puma, Timothy P.Noonan, Paul E. Kennedy, Bruce G. Sturgeon, Bing H. Wang, and Jin-yan Tang The Regulatory Process and Gene Therapy, Suzanne L. Epstein Production of Clinical Lots of Gene Therapy Vectors Using Good Manufacturing Practice: Experience in a University Setting, Alan R. Davis and Colleen Baker Gene Therapy Clinical Trials for Adenosine Deaminase Deficiency/Severe Combined Immunodeficiency, Erlinda M. Gordon and W. French Anderson Development of an Oligodeoxynucleotide Pharmaceutical for the Treatment of Human Leukemia, Alan M. Gewirtz and Deborah Lee Sokol Clinical Trials with Anti-p53 DNA, OL(1)p53, in Patients with Acute Myelogenous Leukemia and Myelodysplastic Syndrome, Patrick L. Iversen Human Bcl-2 Antisense Therapy for Lymphomas, Finbarr E. Cotter, Andrew Webb, Paul Clarke, and David Cunningham Retroviral Gene Transfer in Autologous Bone Marrow and Stem Cell Transplantation, Rafat Abonour and Kenneth Cornetta Adenoviral Gene Transfer of the Herpes Virus Thymidine Kinase Gene for Treating Gliomas, Jane B. Alavi, Jason G. Smith, and Stephen L. Eck Distribution and Toxicity of Retroviral Vectors After Intracavitary Delivery in Mouse and Man, Patrice S. Obermiller, Anne M. Pilaro, Carlos L. Arteaga, and Jeffrey T. Holt Clinical and Immunologic Responses to Gene Transfer of an Allogeneic Major Histocompatibility Complex Antigen, Alison T. Stopeck and Evan M. Hersh Defective Tumor Suppressor Gene Replacement and Oncogene Inactivation for the Treatment of Cancer, Jack Roth The Molecular Basis of Bladder Cancer and Prospects for Gene Therapy Using Hammerhead Ribozymes, Eric J. Small, Mohammed Kashani-Sabet, David Y. Bouffard, and Kevin J. Scanlon In Situ Gene Insertion for Immunotherapy Using Vaccinia Virus Vectors, Edmund C. Lattime, Laurence C. Eisenlohr, and Michael J. Mastrangelo Antisense Oligonucleotide-Based Therapy for HIV-1 Infection from Laboratory to Clinical Trials, Sudhir Agrawal Treatment of Retinitis Induced by Cytomegalovirus Using Intravitreal Fomivirsen (Isis 2922), Stanley T. Crooke Synthetic DNA-Based Compounds for the Prevention of Coronary Restenosis: Current Status and Future Challenges, Andrew Zalewski, Yi Shi, John D. Mannion, and Fernando Roque Prevention of Restenosis by Gene Targeting, Michael J. Mann, Heiko E. Von der Leyen, and Victor J. Dzau

Many RNA species have been identified as important players in the development of chronic diseases, including cancer. Over the past decade, numerous studies have highlighted how regulatory RNAs such as microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) play crucial roles in the development of a disease state. It is clear that the aberrant expression of miRNAs promotes tumor initiation and progression, is linked with cardiac dysfunction, allows for the improper physiological response in maintaining glucose and insulin levels, and can prevent the appropriate integration of neuronal networks, resulting in neurodegenerative disorders. Because of this, there has been a major effort to therapeutically target these noncoding RNAs. In just the past 5 years, over 100 antisense oligonucleotide-based therapies have been tested in phase I clinical trials, a quarter of which have reached phase II/III. Most notable are fomivirsen and mipomersen, which have received FDA approval to treat cytomegalovirus retinitis and high blood cholesterol, respectively. The continued improvement of innovative RNA modifications and delivery entities, such as nanoparticles, will aid in the development of future RNA-based therapeutics for a broader range of chronic diseases. Here we summarize the latest promises and challenges of targeting noncoding RNAs in disease.

Oligonucleotide therapy has come a long way since the early days. Ongoing research is finding more and more applications for this therapeutic tool. At present there are six FDA approved drugs based on oligonucleotide therapy. These are fomivirsen for treatment of CMV retinitis in AIDS patients, mipomersen for treatment of familial hypercholesterolemia, defibrotide for treatment of venoocclusive disease in liver, eteplirsen for treatment of Duchene Muscular Dystrophy, pegaptanib for treatment of neovascular age related macular degeneration and nusinersen for management of spinal muscular atrophy.

Most viral diseases, with the exception of those caused by hu- man immunodeficiency virus, are self-limited illnesses that do not require specific antiviral therapy. The currently available antivi- ral drugs target 3 main groups of viruses: herpes, hepatitis, and influenza viruses. With the exception of the antisense molecule fomivirsen, all antiherpes drugs inhibit viral replication by serving as competitive substrates for viral DNA polymerase. Drugs for the treatment of influenza inhibit the ion channel M 2 protein or the en- zyme neuraminidase. Combination therapy with Interferon-α and ribavirin remains the backbone treatment for chronic hepatitis C; the addition of serine protease inhibitors improves the treatment outcome of patients infected with hepatitis C virus genotype 1. Chronic hepatitis B can be treated with interferon or a combina- tion of nucleos(t)ide analogues. Notably, almost all the nucleos(t) ide analogues for the treatment of chronic hepatitis B possess an- ti-human immunodeficiency virus properties, and they inhibit rep- lication of hepatitis B virus by serving as competitive substrates for its DNA polymerase. Some antiviral drugs possess multiple potential clinical applications, such as ribavirin for the treatment of chronic hepatitis C and respiratory syncytial virus and cidofovir for the treatment of cytomegalovirus and other DNA viruses. Drug resistance is an emerging threat to the clinical utility of antiviral drugs. The major mechanisms for drug resistance are mutations in the viral DNA polymerase gene or in genes that encode for the viral kinases required for the activation of certain drugs such as acyclovir and ganciclovir. Widespread antiviral resistance has lim- ited the clinical utility of M 2 inhibitors for the prevention and treat- ment of influenza infections. This article provides an overview of clinically available antiviral drugs for the primary care physician, with a special focus on pharmacology, clinical uses, and adverse effects.

RNA INTERFERENCE AND ANTISENSE APPROACH There are basically two therapeutic oligonucleotides antisense strategies to inhibit messenger RNA (mRNA). One approach involves using DNA-based single-stranded oligonucleotides referred to as antisense and the other using an RNA-based double-stranded oligonucleotide (dsRNA) known as interference (RNAi). Both entail Watson-Crick binding. Both are oligonucleotides that target specific mRNA for inhibition. Antisense DNA has been in clinical development for some years now with a Food and Drug Administrationapproved product, fomivirsen (intraocular injection), as treatment for CMV retinitis.1 RNAi is only now entering phase I investigation. The existence of double-stranded RNA integral to RNAi was found to exist in nature in 1998.2 RNAi has generated considerable interest as a tool for studying gene function and as a potential therapeutic strategy.3 In addition, it has far greater potency than DNA-based antisense.4 Both strategies offer a readier capacity to be designed against molecular targets compared with small molecules given the ability, in principle, to readily alter or create sequences to match any desired mRNA.

Human cytomegalovirus (HCMV) infection is associated with severe morbidity and mortality in immunocompromised individuals, in particular transplant recipients and AIDS patients, and is the most frequent congenital viral infection in humans. There are currently five drugs approved for HCMV treatment: ganciclovir and its prodrug valganciclovir, foscarnet, cidofovir and fomivirsen. These drugs have provided a major advance in HCMV disease management, but they suffer from poor bioavailability, significant toxicity and limited effectiveness, mainly due to the development of drug resistance. Fortunately, there are several novel and potentially very effective new compounds which are under pre-clinical and clinical evaluation and may address these limitations. This review focuses on HCMV proteins that are directly or indirectly involved in viral DNA replication and represent already established or potential novel antiviral targets, and describes both currently available drugs and new compounds against such protein targets.

Cytomegalovirus (CMV) infection is one of the most important infectious complications of solid-organ transplantation, a serious, life-threatining, opportunistic pathogen in HIV-infected patients, and may cause hearing defects and irreversible central nervous system disease in infants infected during gestation. Four drugs are currently licensed for prophylaxis, pre-emptive therapy, and treatment of CMV infection -- ganciclovir, and its oral prodrug valganciclovir, foscarnet, cidofovir, and fomivirsen. All four drugs are effective against CMV infection. Toxicities, drug-drug interactions, poor bioaailibility, and the development of drug resistance, however, are clinically relevant and common limitations of these drugs. Novel compounds are on the horizon that possibly will become useful alternatives to currently licensed drugs. Maribavir, a benzimidazol, is the most promising novel drug and closest to clinical application. Several phase II clinical trials proved its good tolerability and effectivity. Other compounds are currently evaluated in pre-clinical and phase I trials with promising preliminary data. In addition, analogs of cidofovir posess significantly improved pharmacological and virological characteristics allowing their oral administration. This review summarizes the current status in drug development and will introduce the most recent patents on this line of research.

Oligonucleotide therapeutics are short, single- or double-stranded DNA or RNA molecules consisting of strands of 10-50 nucleotides. By targeted modulation of gene expression oligonucleotides provide the chance of targeting diseases at their molecular level. Within this novel emerging class of compounds oligonucleotide therapeutics are discriminated by their structure, function and mode of action. While antisense oligonucleotides, ribozymes and siRNAs suppress the expression of a protein by complementary hybridizing with their target mRNA, aptamers bind like antibodies to their target protein and thereby inhibit its function. Immunostimulatory oligonucleotides are due to sequence motifs within their nucleotide sequence able to trigger a therapeutic exploitable immune response. Currently, there are only two oligonucleotide therapeutics approved by the FDA, namely the antisense oligonucleotide Fomivirsen and the aptamer Macugen. In this review the mode of action of the diverse oligonucleotide therapeutics and their current status in clinical development will be discussed.

Cytomegalovirus is the most common cause of congenital infection. Congenital cytomegalovirus infection can follow both primary and recurrent maternal infections. It is associated with a significant burden of disease and death. The determinants of mother-to-child transmission and the pathogenesis of symptomatic fetal infection remain poorly understood. For a long time, congenital cytomegalovirus infection has been a neglected disease. Recently, the Institute of Medicine has recognized that the development of a vaccine against congenital cytomegalovirus infection is a public health priority, which should stimulate research in this area. The development of antiviral therapies to prevent symptoms in infected newborns also represents an important area of research.

C YTOMEGALOVIRUS (CMV) RETINITIS IS AMONG THE most common opportunistic infections in patients with the acquired immunodeficiency syndrome (AIDS).1 Before the availability of highly active antiretroviral therapy (HAART), CMV retinitis was estimated to affect 30% of patients with AIDS at some time during the course of their disease.2 Although HAART has been reported to result in 55%–95% decreases in the number of new cases of CMV retinitis,3–5 there appears to have been a leveling off of this decline with the incidence of CMV retinitis at 25% to 30% of its peak incidence.3,6 Although the incidence has declined, as survival among patients with AIDS improves, there is an increasing prevalent population of patients alive with opportunistic infections, such as CMV retinitis. Before the advent of HAART

Under clinical development since the early 90's and with two successfully approved drugs (Fomivirsen and Mipomersen), oligonucleotide-based therapeutics has not yet delivered a clinical drug to the market in the cancer field. Whilst many pre-clinical data has been generated, a lack of understanding still exists on how to efficiently tackle all the different challenges presented for cancer targeting in a clinical setting. Namely, effective drug vectorization, careful choice of target gene or synergistic multi-gene targeting are surely decisive, while caution must be exerted to avoid potential toxic, often misleading off-target-effects. Here a brief overview will be given on the nucleic acid chemistry advances that established oligonucleotide technologies as a promising therapeutic alternative and ongoing cancer related clinical trials. Special attention will be given toward a perspective on the hurdles encountered specifically in the cancer field by this class of therapeutic oligonucleotides and a view on possible avenues for success is presented, with particular focus on the contribution from nanotechnology to the field. The authors would like to acknowledge the FEDER funds through the Programa Operacional Factores de Competitividade - COMPETE and the Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia (PTDC/CTM-NAN/115124/2009, HMSP-ICT/0020/2010 and PEst-C/SAU/LA0002/2013) that supported this work. Pedro M. D. Moreno is supported by a Marie Curie Action of the European Community’s Seventh Framework Program (PIEF-GA2 011300485).

Cytomegalovirus (CMV) infection was recognised in congenitally infected infants in the first half of the 20th century. Following the increased use of immunosuppressive regimens for bone marrow and solid organ transplantation, various manifestations of CMV disease were recognised. Milder symptoms included fever, anorexia and malaise but severe symptoms included pneumonitis, hepatitis, gastrointestinal ulceration, choreoretinitis and encephalopathy, all with a high morbidity or mortality. With the onset of the AIDS epidemic, manifestations of CMV became evident, predominantly retinitis. Ganciclovir used intravenously has been the principal anti-CMV agent investigated. However, ganciclovir has problems with suboptimal efficacy, toxicity, poor oral bioavailability and evolution of resistant strains. Additional studies have been performed on foscarnet and cidofovir, although the use of both have been limited by their nephrotoxicity. Combination therapy with ganciclovir and foscarnet for resistant strains has been used. There are promising newer drugs like the methylenecyclopropane nucleoside analogues and benzimidazole. The most novel compound is the antisense oligonucleotide fomivirsen that has been evaluated principally in CMV retinitis. The role of immunotherapy with either immunoglobulin prophylaxis or the novel adoptive immunotherapy needs further evaluation.

The literature has been reviewed for contact dermatitis from topical antiviral drugs. 15 agents have been identified including acyclovir, imiquimod, podophyllin, podofilox, cidofovir, penciclovir, vidarabine, idoxuridine, trifluridine, tromantadine, lamivudine, interferon intralesional injections and ophthalmic solution, fomivirsen and foscarnet intravitreal injections and ganciclovir intraocular implants. Patch testing has been documented in certain individuals and cross-sensitization has been observed to contribute significantly to some allergic reactions.

Cytomegalovirus (CMV) is a member of the herpesvirus family that is very prevalent world wide based on serologic testing. In immunocompromised persons CMV produces high rates of morbidity and mortality. Congenital CMV is the leading infectious cause of fetal abnormalities in the United States. Infection of human immunodeficiency virus (HIV) seropositive persons or transplant patients with CMV can produce retinitis, encephalitis, pneumonitis, hepatitis, gastrointestinal ulcerations, and cutaneous lesions. Three intravenous therapies are available for CMV infections: ganciclovir; foscarnet and cidofovir. Most recently a fourth antiviral agent was approved for intravitreal injection. This drug, fomivirsen, is the first antisense oligonucleotide available for therapeutic use. A number of other antiviral drugs and vaccines are currently under study.

The introduction of highly active antiretroviral therapy (HAART) for HIV has had a major impact on the treatment of CMV disease in HIV-infected individuals. There is mounting evidence that in patients with CMV retinitis who have a sustained response to HAART, CMV maintenance treatment can be discontinued without relapse of retinitis. In HAART-naïve individuals with newly diagnosed CMV retinitis, the optimal timing for the initiation of HAART relative to the start of anti-CMV treatment is currently unknown. New local therapies for CMV retinitis (e.g. ganciclovir implant, the new antisense compound fomivirsen) provide treatment options in situations where high local drug delivery is warranted. A treatment algorithm for CMV disease in the HAART era is proposed. In the transplant setting, ganciclovir and foscarnet remain the major compounds used for treatment of CMV disease. In marrow and stem cell transplant recipients, CMV pneumonia still carries a high mortality. Ganciclovir in combination with CMV-specific immunoglobulin or regular intravenous IG remains the treatment of choice for CMV pneumonia; extended antiviral maintenance for several months is recommended in patients with continued immunosuppression. Preemptive treatment based on virologic markers (e.g. pp65 antigenemia, CMV DNA) has been very successful in reducing the incidence of early CMV disease in the transplant setting. The duration of preemptive treatment should be guided by the underlying immunosuppression and virologic markers. Late CMV disease is a challenge in marrow and stem cell transplant recipients, and occurs increasingly in highly immunosuppressed solid organ transplant recipients as well. Recent advances in prophylaxis strategies include oral ganciclovir for liver transplant recipients and valacyclovir for kidney transplant recipients.

Background CMV retinitis is the most common manifestation of active CMV infection in patients with AIDS. Before ganciclovir became available, the prognosis of CMV retinitis was very poor. Meanwhile a number of strategies for the management of CMV retinitis have evolved. To date no antiviral agent has proved effective in preventing reactivation of retinitis. Therefore the available antiviral agents delay rather than prevent relapse of CMV retinitis. Each relapse produces a more serious disease and retinitis becomes progressively less manageable. Method A review of current therapeutic strategies for the treatment of CMV retinitis with regard to the challenge of clinical resistance is given. Conclusion Effective treatment of clinically resistant CMV retinitis must consider the individual conditions. Thus, it is probable, that integration of both local and systemic therapies may be required to reduce the frequency of reactivation and thereby to decelerate the progression of the disease. Literature search in medline.

The field of antisense oligonucleotides (ASONs) has now matured to the point that much of the original optimism regarding their therapeutic potential has been rekindled. The first antisense to reach the commercial stage, fomivirsen (Vitravene™) for cytomegalovirus (CMV) retinitis in immunocompromised patents, may be prescient for future successful members of this new therapeutic class in that it clearly illustrates the utility of ASONs when the problem of delivery is solved. The simplest way to solve the delivery problem is to administer ASONs directly, i.e., locally, to the target tissue. Fomivirsen is approved for direct intravitreal injection. This brief review examines recent work on local delivery of ASONs to treat a variety of human diseases.

Fomivirsen is a 21-nucleotide phosphorothioate oligonucleotide which, when injected into a human eye, is capable of inhibiting CMV retinitis. Its mode of action is consistent with an antisense mechanism. Prior to human trials, fomivirsen was tested in a number of in vitro cell lines and was found to inhibit CMV replication in a dose-dependent manner with a mean 50% inhibitory concentration between 0.03 and 0.2 microM. Intravitreal drug clearance studies have revealed first-order kinetics with a half-life in the rabbit of 62 hours. In a clinical trial of patients with newly diagnosed CMV retinitis receiving 165 mg per injection, time to progression was interpolated to 71 days with 44% of the patients remaining on treatment for over one year. In patients who failed other anti-CMV treatments, the interpolated time to progression was 91 days when receiving 330 mg per injection. No systemic absorption of the drug could be detected. Reported adverse events have been for the most part mild to moderate in intensity and either resolved spontaneously or were treatable with topical medications. Locally administered fomivirsen effectively inhibits CMV retinitis using a mode of action which is complementary to existing DNA polymerase inhibitors.

Background: With the advent of nucleic acids as therapeutic molecules, the traditional drug discovery and development process has undergone a radical change. Because nucleic acids target complementary sequences and the recognition is on the basis of Watson–Crick base pair formation, the specificity of the whole process is high. The unwanted side reactions, which are a common phenomenon observed with small molecules, are thus cut down. Objectives: The objective of this review is to look at the concept of antisense oligonucleotides as nucleic acid medicines and trace the history of drug molecules based on this concept that are at various stages of advancement. The problems encountered in the development process and the possible delivery routes are critically analyzed. Conclusion: Although specificity and selectivity are the key features of antisense oligonucleotides, the need to target the right tissues and reach the nucleus remains a challenge to overcome.

Since the first pioneering studies using antisense oligonucleotides (ASOs) in the late 1970s, thousands of publications followed, demonstrating the remarkableness of antisense action and its enormous application spectrum. In 1998, Fomivirsen (Vitravene) was the first, and to date the only ASO that gained approval by the US Food and Drug Administration (FDA) for intravitreous treatment of cytomegalovirus-induced retinitis in patients with acquired immune deficiency syndrome (AIDS). Meanwhile, efforts regarding ASO research decreased and investigations shifted to other molecules, e.g., small interfering RNAs, because ASO-related problems such as insufficient efficacy and off-target effects are not yet overcome. However, newer studies using ASOs with improved chemistry or approaches combining ASO treatment with other therapies, such as chemotherapy or radiation, might bring ASOs back into the spotlight. This chapter will focus on current in vivo studies and clinical trials of promising ASOs.