Lentiviral vectors

The delivery of genetic material to mammalian cells is of great importance for modern fundamental biology, biomedicine, biotechnology, agriculture, and veterinary medicine. The development of new efficient techniques of gene transfer to human cells led to the advent of gene therapy, a novel approach to treating severe metabolic disorders, some viral infections (including HIV infection), autoimmune diseases, and genetic defects causing cancer. The review considers the main principles of constructing gene transfer and expression systems based on lentiviruses, a powerful tool for human gene therapy and transgenic research, with a special focus on the genome structure and life cycle of lentiviruses and the design and safety of lentiviral vector systems.     

Lentiviral vectors

The delivery of genetic material to mammalian cells has a great importance for modern fundamental biology, biomedicine, biotechnology, agriculture and veterinary medicine. The development of new efficient techniques of gene transfer to human cells has led to the establishment of gene therapy, a novel type of treatment targeting severe metabolic disorders, some viral infections, including HIV, autoimmune diseases and genetic defects causing cancer. This review summarizes the achievements in lentiviral-mediated gene transfer, a powerful tool for use in human gene therapy and transgenic research, with a special focus on the genome structure and life cycle of lentiviruses, as well as on the design and safety aspects of lentiviral vector systems.     

Retroviral vectors

The majority of clinical trials for gene therapy currently employ retroviral-mediated gene delivery. This is because the life cycle of the retrovirus is well understood and can be effectively manipulated to generate vectors that can be efficiently and safely packaged. Here, we review the molecular technology behind the generation of recombinant retroviral vectors. We also highlight the problems associated with the use of these viruses as gene therapy vehicles and discuss future developments that will be necessary to maintain retroviral vectors at the forefront of gene transfer technology.     

Lentiviral vectors

Vectors based on lentiviruses have reached a state of development such that clinical studies using these agents as gene delivery vehicles have now begun. They have particular advantages for certain in vitro and in vivo applications especially the unique capability of integrating genetic material into the genome of non-dividing cells. Their rapid progress into clinical use reflects in part the huge body of knowledge which has accumulated about HIV in the last 20 years. Despite this, many aspects of viral assembly on which the success of these vectors depends are rather poorly understood. Sufficient is known however to be able to produce a safe and reproducible high titre vector preparation for effective transduction of growth-arrested tissues such as neural tissue, muscle and liver.     

Q vectors, bicistronic retroviral vectors for gene transfer

We have developed a retroviral vector that incorporates unique features of some previously described vectors. This vector includes: 3' long terminal repeats (LTRs) of the self-inactivating class; a 5' LTR that is a hybrid of the cytomegalovirus (CMV) enhancer and the mouse sarcoma virus promoter; an internal CMV immediate early region promoter to drive expression of the transduced gene and the neomycin phosphotransferase selectable marker; an expanded multiple cloning site and an internal ribosome entry site. An SV40 ori was introduced into the vector backbone to promote high copy number replication in packaging cell lines that express the SV40 large T antigen. We demonstrate that these retroviral constructs, designated Q vectors, can be used in applications where high viral titers and high level stable or transient gene expression are desirable.     

Autonomous parvovirus vectors

Parvoviruses are small, icosahedral viruses (approximately 25 nm) containing a single-strand DNA genome (approximately 5 kb) with hairpin termini. Autonomous parvoviruses (APVs) are found in many species; they do not require a helper virus for replication but they do require proliferating cells (S-phase functions) and, in some cases, tissue-specific factors. APVs can protect animals from spontaneous or experimental tumors, leading to consideration of these viruses, and vectors derived from them, as anticancer agents. Vector development has focused on three rodent APVs that can infect human cells, namely, LuIII, MVM, and H1. LuIII-based vectors with complete replacement of the viral coding sequences can direct transient or persistent expression of transgenes in cell culture. MVM-based and H1-based vectors with substitution of transgenes for the viral capsid sequences retain viral nonstructural (NS) coding sequences and express the NS1 protein. The latter serves to amplify the vector genome in target cells, potentially contributing to antitumor activity. APV vectors have packaging capacity for foreign DNA of approximately 4.8 kb, a limit that probably cannot be exceeded by more than a few percent. LuIII vectors can be pseudotyped with capsid proteins from related APVs, a promising strategy for controlling tissue tropism and circumventing immune responses to repeated administration. Initial success has been achieved in targeting such a pseudotyped vector by genetic modification of the capsid. Subject to advances in production and purification methods, APV vectors have potential as gene transfer agents for experimental and therapeutic use, particularly for cancer therapy.     

Targeted adenoviral vectors

Replication-defective vectors based on human adenovirus serotypes 2 and 5 (Ad2 and Ad5) possess a number of attributes which favor their use as gene delivery vehicles in gene therapy applications. However, the widespread distribution of the primary cellular receptor for Ad, the coxsackievirus and adenovirus receptor (CAR), allows Ad vectors to infect a broad range of cells in the host. Conversely, a number of tissues which represent important targets for gene therapy, such as the airway epithelium and cancer cells, are refractory to Ad infection due a paucity of CAR. Thus, there is a strong rationale for the development of CAR-independent Ad vectors capable of enhanced specificity and efficiency of gene transfer to target cells. In this article we review the approaches which have been employed to generate tropism-modified Ad vectors. These targeting strategies have led to improvements in the safety and efficacy of Ad vectors and have the potential to yield an increased therapeutic benefit in the human clinical context.     

Specific-purpose broad-host-range vectors

Several plasmid derivatives of broad-host-range Inc P4 plasmid RSF1010 were constructed and characterized. Vector pAYC30 was constructed by insertion in vivo into the genome of RSF1010 the Hgr transposon Tn501, originating from the plasmid pVS1 of Pseudomonas aeruginosa. Plasmids with inserts of PstI or SacI fragments may be selected by inactivation of genes sul and aph, respectively. The cloning at unique site SalGI leads to the appearance of HgCl2--sensitive transformants. Versatile cloning vector pAYC1 consists of two replicons, RSF1010 and plasmid pMZ7, a derivative of R6K. The constructed plasmid is 16.9 kb in length and determines resistance to five drugs. Two promoter-probe broad-host-range vectors, pAYC36 and pAYC37, were obtained by replacing a small segment from the DNA sequence of the aph gene promoter of previously described plasmid pAYC32 with the polylinker from plasmid pUC19. Therefore, vector plasmids retained the intact gene aph (Smr); however, they have Sms phenotype because of the insertional inactivation of the promoter. The genetic structure of promoter-probe vectors allows one to select clones, containing hybrid plasmids with an active promoter for gene aph expression.     

Positive selection vectors

This review describes information concerning positive selection vectors on their mechanism, classification, property, and limitation. A total of 72 positive selection vectors collected were discussed. Positive selection vectors can reduce background and directly screen transformants containing cloned DNA fragments. The mechanisms to perform positive selection include insertional inactivation and the replacement of functional genes of the vectors. In general, the former is much more convenient than the latter. The functional genes are controlled either by their promoters or by heterologous promoters introduced. On the basis of the structures, positive selection vectors could be classified into five groups. The positive selection vectors are commonly based on the mechanisms of lethal genes and the sensitivity of compounds. The vectors, with molecular weights ranging from 2.6 to 17.0 kb, have diverse genetic markers and wide host ranges, including Escherichia coli, Bacillus, Streptomyces, lactic acid bacteria, yeasts, and mammalian cells. Although some limitations exist for using some positive selection vectors, they are useful in recombinant DNA experiments.     

Improved EBV shuttle vectors

Shuttle vectors based on Epstein-Barr virus (EBV) replicate autonomously in the nuclei of human cells. These vectors represent reasonable models for chromosomes, have low background mutation frequencies, and have been useful for studying induced mutation in human cells. Two improvements in the EBV vector system are discussed. Attempts are described to increase vector copy number per cell by using a limited period of replication driven by the simian virus 40 (SV40) origin of replication. Isolation of human sequences that can replace the viral origin of replication in providing for autonomous replication of the vectors is also described. These improvements are leading toward shuttle vectors that are more efficient and more closely resemble authentic chromosomes.