**3. Autonomous parvovirus vectors**

Autonomously replicating parvovirus (ARP) can replicate in proliferating cells without the need for a helper virus. This is one feature that makes the ARPs attractive for potential vector production. ARPs 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 [38]. Most vector work has been focused on autonomous parvoviruses that can infect human cells, namely, LuIII, MVM (minute virus of mice), and H1, which are members of the rodent group of APVs. Autonomous parvoviruses were first isolated from human tumor tissue and it was then observed that they possess an onco-suppressive potential, inhibiting the formation of spontaneous and chemically or virally induced tumors in vivo and in vitro [39–41]. Autonomous parvoviruses express preferentially in cancerous cells and possess oncolytic activity that has led to their implication in potential use as vectors for cancer gene therapy. Moreover, these viruses do not cause pathogenicity in adult animals and they seem to be associated with low or no immunogenicity. Additional features that make APVs interesting candidates for gene therapy are their episomal replication and high stability [42]. 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.

The genome of ARP comprises of two nonstructural proteins and viral capsid proteins. The non-structural proteins, NS-1 and NS-2 are highly conserved among the rodent parvoviruses that lead to cross-reactivity in serological assays utilizing whole virus antigen. The viral capsid proteins, VP-1 and VP-2 are specific to the virus and form the basis for serological differentiation. All currently proposed MVM and H-1 vectors retain the palindromes and NS1-coding sequences [42]. Other than its role in viral DNA replication, NS1 also possess the cytotoxic activity in tumor cells which should contribute to the destruction of tumor cells directly, through oncolysis, and indirectly through the induction of an immune response via the presentation of tumor-associated antigens by APCs to lymphocytes [43]. In addition, the late promoter P38 that regulates the expression of capsid proteins is transactivated by NS1. This promoter is used for the expression of transgenes to ensure that their expression occurs only in cells that also express NS1. VP proteins are generally expressed from P38, either on a helper plasmid or in packaging cells, thereby linking the expression of capsid proteins to the viral life cycle [43].

The major problems encountered with these vectors are their low titers and the generation of wild-type or replication-competent virus (RCV) through recombination with helper plasmids [42]. Over the time, advancements have been made to enhance the titers of recombinant virus and to reduce the contamination by RCV. Genetic engineering of vector has led to their enhanced production after transfection [44]. The reduction of homology between vector and helper sequences as well as integration of helper sequences into host cell genomes have greatly reduced the generation of RCV [45, 46].

Similarly, production of LuIII transducing virus has been accomplished by co-transfection of plasmid-based helper and transducing genome constructs [47]. In general, during co-transfections to generate transducing virus, recombination between helper and transducing genomes can regenerate infectious virus with variable frequencies [47]. Elimination of DNA-DNA recombination can be achieved by providing one of the components of the packaging system in RNA form. Sindbis, the plus-strand RNA virus that can express large amounts of protein from foreign genes in a variety of vertebrate cell types [48], were used for providing components of the LuIII packaging system in RNA form. Sindbis replicon vector was used to express NS1, the major non-structural protein of LuIII. Sindbis-expressed NS1 RNA and protein were readily detectable in cultured cells; this NS1 was able to mediate production of LuIII-luciferase transducing virus [48].
