Design and delivery of non-parenteral vaccines

Non-parenteral delivery of vaccines is reviewed focusing on the delivery systems that have been used for various mucosal routes of administration. Systems considered include biodegradable micro- and nanoparticles, liposomes, live bacterial and viral vectors and mucosal adjuvants. New approaches to mucosal vaccine formulation using: (i) gene fusion technology to create non-toxic derivatives of mucosal adjuvants, (ii) genetically inactivated antigens with a deletion in an essential gene, (iii) coexpression of an antigen and a specific cytokine that is important in the modulation and control of a mucosal immune response, and (iv) genetic material itself that would allow DNA or RNA uptake and its endogenous expression in the host cell are described.     

The prospects for gene therapy in cystic fibrosis

Gene therapy provides the best prospect of a fundamental new treatment for cystic fibrosis. The lungs are the most important target, because this organ is the most severely affected by the disease and is also accessible for topical treatment. Advances in this field have been very rapid, and the prospects remain good although a number of problems need to be overcome. The two main approaches to gene transfer, namely adenoviruses and liposomes, are efficient in vitro, but early clinical trials have shown that they work less well in vivo. A number of proof of concept studies have shown that gene transfer is possible, but full functional correction of the cystic fibrosis defect has not yet been achieved. Adenoviruses have provoked an inflammatory response, and new viral vectors are being developed to overcome this. Existing lipids are relatively inefficient, but new liposomes are being developed to enhance gene transfer. Much work needs to be done to improve safety and efficacy of gene transfer before materials are ready for large scale clinical trials. However, progress is very rapid, and there is a real prospect of developing an effective gene therapy for cystic fibrosis within the next decade.     

Cellular and molecular neurosurgery: pathways from concept to reality--part I: target disorders and concept approaches to gene therapy of the central nervous system

Different vector systems that have been used and/or specifically developed for central nervous system (CNS) gene transfer studies are briefly discussed along with their advantages and disadvantages with respect to potential clinical application. These include retroviruses, recombinant herpes simplex virus, adenoviruses, adenoassociated viruses, encapsulation of plasmid deoxyribonucleic acid into cationic liposomes, and neural and oliogodendroglial stem cells. Particular attention has been paid to relate the modality of a specific CNS gene therapy to the strategy for adequate delivery of genetic material to the brain for either global or localized CNS neurodegenerative chronic disorder, as well as for CNS tumors and stroke. Techniques to circumvent the "impermeable" blood-brain barrier and how to breach the more versatile blood-brain-tumor barrier to deliver the genetic material to the target CNS cells are reviewed and include the following: 1) local stereotactic CNS injection/infusion of viral vectors, administration of vector producer cells, or cell replacement; 2) local administration of genetic material into the cerebrospinal fluid ventriculocisternal system; 3) osmotic opening of the blood-brain barrier; 4) local intra-arterial infusion; and 5) administration of blood-brain-tumor barrier permeabilizers, such as a bradykinin B2 agonist RMP-7. It is concluded that gene therapy for several brain disorders holds great potential, as suggested mainly by in vitro experiments and, to some extent, by a limited number of animal experiments. However, several drawbacks currently hamper the application of gene therapy under the clinical setting. The problems associated with gene therapy that still present major obstacles are as follows: 1) inefficient transfection of host cells by viral vectors; 2) restricted delivery of genetic material across vascular barriers of the CNS and brain tumors; 3) nonselective expression of the transgene; and 4) in situ CNS regulation of the transgene expression in a therapeutically controlled manner, as imposed by the course and phenotype of the CNS disease.     

Muscle maturation: implications for gene therapy

Skeletal muscle is a promising target tissue for gene therapy, for both muscle and non-muscle disorders. A variety of methods have been studied to transfer genes into skeletal muscle, including retroviral, adenoviral and herpes simplex viral vectors. However, various factors impede muscle-based viral gene therapy. Here, we discuss why some viral vectors cannot efficiently transduce mature muscle fibers, and describe some new approaches to overcome this barrier.     

Gene transfer by guanidinium-cholesterol cationic lipids into airway epithelial cells in vitro and in vivo

Synthetic vectors represent an attractive alternative approach to viral vectors for gene transfer, in particular into airway epithelial cells for lung-directed gene therapy for cystic fibrosis. Having recently found that guanidinium-cholesterol cationic lipids are efficient reagents for gene transfer into mammalian cell lines in vitro, we have investigated their use for gene delivery into primary airway epithelial cells in vitro and in vivo. The results obtained indicate that the lipid bis(guanidinium)-tren-cholesterol (BGTC) can be used to transfer a reporter gene into primary human airway epithelial cells in culture. Furthermore, liposomes composed of BGTC and dioleoyl phosphatidylethanolamine (DOPE) are efficient for gene delivery to the mouse airway epithelium in vivo. Transfected cells were detected both in the surface epithelium and in submucosal glands. In addition, the transfection efficiency of BGTC/DOPE liposomes in vivo was quantitatively assessed by using the luciferase reporter gene system.     

Three-Dimensional Reconstruction of a Collagen IV Analogue in the Dogfish Egg Case Wall

Autonomously replicating virus-based vectors have been investigated as a means of introducing heterologous genes into plants. This approach has a number of potential advantages over stable genetic transformation, particularly in terms of speed and levels of expression that can be obtained. Several groups of plant viruses, with genomes consisting of both DNA and RNA, have been investigated as possible gene vectors. In the case of DNA viruses, it has generally been possible to identify nonessential regions of the genome that can be replaced by foreign sequences. However, there appear to be limitations on the size of insert which can be tolerated. In the case of RNA viruses, replacement of viral sequences usually has a drastic effect on the viability. However, in several cases it has proved possible to substantially increase the size of the viral genome by the direct insertion of additional sequences while still retaining the ability of the viruses to multiply and spread in plants. These RNA virus-based systems appear to have the greatest potential as gene vectors.     

Gene therapy for cerebrovascular disease

OBJECTIVE: To review the principles of and the experimental and clinical results of gene therapy for cerebrovascular disease. METHODS: Literature review. RESULTS: Vectors for gene transfer into the brain or into the cerebral vasculature include naked plasmid deoxyribonucleic acid, cationic liposomes, and viruses such as adenovirus, retrovirus, adeno-associated virus, and herpes simplex virus. Experiments using these vectors showed that intra- or perivascular application to systemic arteries can lead to transfection and expression of a foreign transgene in the adventitia and the endothelium. Intrathecal administration can lead to transfection and foreign transgene expression in leptomeningeal cells as well as in fibroblasts of blood vessel adventitia. Biological effects demonstrated thus far include increased nitric oxide production by transfection of cerebral arterial adventitia with adenovirus expressing nitric oxide synthase. Adenoviruses carrying foreign genes have been used to decrease neuronal damage in cerebral ischemia and to decrease blood pressure in spontaneously hypertensive rats. Vectors and therapeutic applications for gene therapy are evolving rapidly. CONCLUSION: Gene therapy for cerebrovascular disease is likely to have clinical application in the near future and will have a major impact on neurosurgery. Neurosurgeons will need to be aware of the literature in this area.     

An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3

Human adenoviruses (Ads) are attracting considerable attention because of their potential utility for gene transfer and gene therapy, for development of live viral vectored vaccines, and for protein expression in mammalian cells. Engineering Ad vectors for these applications requires a variety of reagents in the form of Ads and bacterial plasmids containing viral DNA sequences and requires different strategies for construction of vectors for different purposes. To simplify Ad vector construction and develop a procedure with maximum flexibility, efficiency, and cloning capacity, we have developed a vector system based on use of Ad5 DNA sequences cloned in bacterial plasmids. Expanded deletions in early region 1 (3180 bp) and early region 3 (2690 or 3132 bp) can be combined in a single vector that should have a capacity for inserts of up to 8.3 kb, enough to accommodate the majority of cDNAs encoding proteins with regulatory elements. Genes can be inserted into either early region 1 or 3 or both and mutations or deletions can be readily introduced elsewhere in the viral genome. To illustrate the flexibility of the system, we have introduced a wild-type early region 3 into the vectors, and to illustrate the high capacity for inserts, we have isolated a vector with two genes totaling 7.8 kb.     

Transportation of cytotoxic liposomes to malignant cells using a carbohydrate determinant

A method of the synthesis of lipophilic glycoconjugates (vectors) on the basis of polyethyleneglycol-containing detergent was proposed. It has been shown by flow cytofluorometry that fluorescent labeled liposomes equipped with beta-galactosyl conjugate are bound human leukosis HL-60 cells more effectively than liposomes embedded with the beta-glucosyl conjugate or vector-free liposomes. A new lipid derivative of antitumor drug rubomycin (daunorubicin), N-(rac-1,2-dioleoylglycero-3-oxalyl)rubomycin (RubDG) has been synthesized. Liposomes loaded with RubDG and equipped with galactosyl vector showed higher cytotoxic activity in vitro against HL-60 cells than analogous unvectored liposomes or liposomes bearing glucosyl conjugate.     

New approaches to the development of virus vaccines for veterinary use

The marked progress in recombinant deoxyribonucleic acid (DNA) technology during the past decade has led to the development of a variety of safe new vaccine vectors which are capable of efficiently expressing foreign immunogens. These have been based on a variety of virus types--poxviruses, herpesviruses and adenoviruses--and have led to the production of many new potential recombinant vaccines. Of these recombinant vaccines, the rabies vaccine, in which the rabies G protein is expressed in a vaccinia vector, has been widely used in the field to prevent the spread of rabies both in Europe and in the United States of America. A recombinant Newcastle disease virus vaccine, using fowlpox virus as the vector to express immunogenic proteins from the Newcastle disease virus, has been licensed as the first commercial recombinant vectored vaccine. Many other recombinant virus vaccines are still at the stage of laboratory or field testing. The most recent breakthrough in vaccinology has been the success with the use of naked DNA as a means of vaccination. This approach has shown great promise in mouse model systems and has now become the most active field in new vaccine development. Molecular redesigning of conventional ribonucleic acid (RNA) viruses to obtain more stable attenuated vaccines was previously possible only for positive-strand RNA viruses, such as poliovirus. However, recent advances in molecular biological techniques have enabled the rescuing of negative-strand viruses from DNA copies of their genomes. This has made it possible to engineer specific changes in the genomes of Rhabdoviridae and Paramyxoviridae, both of which include several viruses of veterinary importance. The authors describe the current progress in the development of vector vaccines, DNA vaccines and vaccines based on engineered positive- and negative-strand RNA virus genomes, with special emphasis on their application to diseases of veterinary importance.     

Manipulation of gene expression in the mammalian nervous system: application in the study of neurite outgrowth and neuroregeneration-related proteins

A fundamental issue in neurobiology entails the study of the formation of neuronal connections and their potential to regenerate following injury. In recent years, an expanding number of gene families has been identified involved in different aspects of neurite outgrowth and regeneration. These include neurotrophic factors, cell-adhesion molecules, growth-associated proteins, cytoskeletal proteins and chemorepulsive proteins. Genetic manipulation technology (transgenic mice, knockout mice, viral vectors and antisense oligonucleotides) has been instrumental in defining the function of these neurite outgrowth-related proteins. The aim of this paper is to provide an overview of the above-mentioned four approaches to manipulate gene expression in vivo and to discuss the progress that has been made using this technology in helping to understand the molecular mechanisms that regulate neurite outgrowth. We will show that work with transgenic mice and knockout mice has contributed significantly to the dissection of the function of several proteins with a key role in neurite outgrowth and neuronal survival. Recently developed viral vectors for gene transfer in postmitotic neurons have opened up new avenues to analyze the function of a protein following local expression in naive adult rodents. The initial results with viral vector-based gene transfer provide a conceptual framework for further studies on genetic therapy of neuroregeneration and neurodegenerative diseases.     

Gene delivery by negatively charged ternary complexes of DNA, cationic liposomes and transferrin or fusigenic peptides

Potential problems with the use of viral vectors for gene therapy necessitate the development of efficient nonviral vectors. The association of transferrin, or the pH-sensitive peptide GALA, with cationic liposomes composed of 1,2-dioleoyl-3-(trimethylammonium) propane and its equimolar mixture with dioleoylphosphatidylethanolamine, under conditions where the liposome/DNA complex is negatively charged, drastically increased luciferase expression from pCMVluc. The percentage of cells transfected, measured by beta-galactosidase expression, was also increased by about 10-fold. The zeta potential of the ternary complexes was lower than that of the liposome/DNA complexes. Transfection activity of positively charged complexes was also enhanced by association with transferrin, GALA or the influenza hemagglutinin N terminal peptide HA-2, but to a smaller extent compared with the negatively charged complexes. The enhancement of gene delivery by transferrin or GALA was not affected significantly by the presence of serum and did not cause significant cytotoxicity. Our results indicate that negatively charged ternary complexes of cationic liposomes, DNA and transferrin, or fusigenic peptides, can facilitate efficient transfection of cultured cells, and that they may alleviate the drawbacks of the use of highly positively charged complexes for gene delivery in vivo.     

Cardiovascular molecular genetics and gene therapy. Introduction to the terminology and technique

The possible application of somatic gene therapy to the treatment of cardiovascular diseases is introduced; methods and delivery systems for transferring genes to the sites of application are discussed. Although cardiovascular diseases are mainly systemic disorders, critical lesions are frequently located at specific sites in the circulation and therefore accessible to the introduction of recombinant genes which may encode for proteins either inhibiting cell proliferation or promoting cell division. Cell-mediated gene transfer is compared to direct gene transfer in vivo; the advantages and disadvantages of the "ex vivo-in vitro-in vivo" and "direct in vivo" techniques are described. Several alternative gene transfer techniques are known. They include viral and retroviral vectors which have been made replication defective by genetic engineering, as well as non-viral systems such as DNA-liposome complexes, and physical methods such as the so-called biolistic technology originally developed to penetrate plant cell membranes. In summary, it is pointed out that even demonstrations of the few successful therapeutic applications to date do not conceal the fact that intensive experimental research using animal models is needed to prove the advantages and safety of the techniques used in gene therapy of the cardiovascular system.     

Analysis of events leading to neuronal death after infection with E1-deficient adenoviral vectors

Although recombinant adenoviral vectors are being widely used to target genes to the nervous system, the cellular and genetic effects of recombinant adenoviral infection on neuronal function have not been well characterized. Using sympathetic neuronal cultures, we analyzed the effect of adenoviral infection on viral and neuronal gene expression and on neuronal function and viability. While a delayed cytotoxicity occurred 5 days after infection, numerous biochemical and genetic perturbations occurred within the infected cell prior to this time. This study demonstrates that numerous cellular alterations were produced by recombinant adenoviral vectors and, therefore, emphasizes the need for an analysis of the effects of these viral vectors on neuronal function in the interpretation of data regarding transgene expression induced by these vectors in neurons. It also suggests that continued improvements made to the viral vectors themselves might decrease this direct cytotoxicity and lead to improved safety and function of recombinant adenovirus in vivo.     

High doses of a helper-dependent adenoviral vector yield supraphysiological levels of alpha1-antitrypsin with negligible toxicity

Optimal gene therapy for many disorders will require efficient transfer to cells in vivo, high-level and long-term expression, and tissue-specific regulation, all in the absence of significant toxicity or inflammatory responses. While recombinant adenoviral vectors are efficient for gene transfer to hepatocytes, their usefulness is limited by short duration of expression related, at least in part, to immune responses to viral proteins and by a low capacity for foreign DNA. A number of systems have been developed for producing adenoviral vectors devoid of all viral coding sequences. Using AdSTK109, a vector lacking all viral coding sequences and carrying the complete human alpha1-antitrypsin (hAAT) genomic DNA locus, we have demonstrated sustained expression for longer than 10 months in mice. Utilizing high doses of this vector for hepatic gene transfer in mice, we find that supraphysiological levels of hAAT can be achieved without hepatotoxicity.     

DNA vaccines

DNA vaccination against infectious diseases has created a new field of applied molecular immunology. cDNAs for 'protective' protein epitopes can be inserted into vectors containing strong mammalian promoters for high expression. Here we discuss the mechanisms of DNA vaccination and the successful and sometimes unsuccessful applications of DNA vaccination to protect animals against many different viral, bacterial mycoplasmal, protozoal, and worm infections or infestations. DNA immunization has been used to prevent or inhibit tumor development and to inhibit IgE responses by diverting the immune response from Th2 to Th1 helper cell dominance. Advantages and disadvantages of a variety of routes of administration and methods of immunization discussed include the use of the 'gene gun', the delivery of genes by aerosols, and deliberate induction of injury to muscles prior to injection of DNA to enhance gene expression. Vaccination performed using DNA without knowing beforehand the protective epitopes, using 'expression library immunization', is discussed. While this field is bound to expand rapidly for future clinical applications, we try to point out potential pitfalls as well as advantages of this relatively new technology.     

Effect of the E4 region on the persistence of transgene expression from adenovirus vectors

The utility of adenovirus vectors for gene therapy is limited by the transience of expression that has been observed in various in vivo models. Immunological responses to viral targets can eliminate transduced cells and cause the loss of transgene expression. We previously described the characterization of an E4 modified adenovirus, Ad2E4ORF6, which is replication defective in cotton rats. We reasoned that gene transfer vectors based on Ad2E4ORF6 would have a reduced potential for viral gene expression in vivo which might be beneficial for achieving persistence of transgene expression. E1 replacement vectors expressing the cystic fibrosis transmembrane regulator or beta-galactosidase were constructed as series of vectors that differed with respect to the E4 region. Vectors containing a wild-type E4 region, E4 open reading frame 6, or a complete E4 deletion were compared in the lungs of BALB/c mice for persistence of expression. Results obtained with nude mice indicate that nonimmunological factors have a major influence on the longevity of transgene expression. Expression was transient from the E1a promoter with all vectors but persisted from the cytomegalovirus promoter only with a vector containing a wild-type E4 region. Transience of expression did not correlate with the disappearance of vector DNA, suggesting that promoter down-regulation may be involved. Coinfection studies indicate an E4 product(s) could be supplied in trans to allow persistent expression from the cytomegalovirus promoter. In summary, the choice of promoter is important for achieving persistence of expression; in addition, some promoters are highly influenced by the context of the vector backbone.     

A simplified vaccinologists' vaccinology and the pursuit of a vaccine against AIDS

Vaccinology is the science and engineering of developing vaccines to prevent infectious diseases. Guidelines come from knowledge of pathogenesis and from successful past vaccines. The vaccine enterprise relies on the evolution of appropriate science and technology. Governmental support and industrial participation are key to successful development of new vaccines. A large challenge for vaccinology is a vaccine which protects against AIDS. Though misguided in its first decade, current vaccine research is directed to use of any and all viral antigens and to elicit both cell-mediated and humoral immune responses that are resident, with memory, at the mucosal sites of viral entry. Recent seminal discoveries guiding the future include selective elicitation of both Type 1 and Type 2 immune responses, and prime-boosting using recombinant viral or DNA vectors and expressed antigens. Success in vaccinology depends on simplification of the complex and on iterative processes in a well-defined pathway. The present and future of vaccinology are discussed in depth.