Vectors vaccinia

Other types of replication-deficient viral vectors that have been used in the gene therapy field include Herpes simplex viral (HSV) vectors that (1) are able to transduce nondividing cells and (2) are highly infective for neurologic tissue and vaccinia vectors. Vaccinia vectors in turn (1) are able to transduce nondividing cells and (2) have the ability to efficiently infect many types of cells. The primary safety concerns for HSV vectors are the potential for tropism to the CNS and the potential for latency and reactivation. Vaccinia vectors contain the same backbone as the smallpox vaccine, thus the available safety databases for vaccinia administration in humans consist primarily of preventive vaccination in a healthy population. Principal safety concerns with the use of vaccinia vectors include (1) their ability to replicate in humans and possibly... 

Live vectors (131,133) are another appHcation of genetic engineering. In this case, the genes from a pathogen are inserted into a vaccine vector, such as salmonella or vaccinia. In the case of salmonella, it will be possible to develop an oral vaccine. Vectors for this appHcation include salmoneUa, BCG, poHo, adenovims, and vaccinia. 

Figure 4.4. Schematic illustration of directional topoisomerase cloning of PCR products into the pUNI vector. The PCR product to be cloned has the sequence 5 -CACC appended at the 5 end to direct the orientation of cloning. The Vaccinia virus topoisomerase I enzyme forms a covalent adduct with the cloning vector to create a cloning competent plasmid construct. The loxP site is 5 to the insertion site. The vector and PCR product are designed to fuse the ORF in-frame with loxP.

A number of factors render vaccinia virus a particularly attractive vector system. These include ... 

Figure 14.2 Vectors used thus far in gene therapy trials. Others are mainly viral-based and include the use of pox, vaccinia and adeno-associated viruses, as well as herpes simplex virus. Data adapted from www.wiley. co.uk/genemed/clinical...

Gene Therapies. The types of vectors that have been used or proposed for gene transduction include retrovirus, adenovirus, adeno-associated viruses, other viruses (e.g., herpes, vaccinia, etc.), and plasmid DNA. Methods for gene introduction include ex vivo replacement, drug delivery, marker studies, and others and in vivo, viral vectors, plasmid vectors, and vector producer cells. 

Viral Vectors. The direct administration of a viral vector (e.g., retrovirus, adenovirus, adeno-associated virus, herpes, vaccinia) to patients. 

Introduction of a gene of interest into the host cell line by viral infection is a convenient method since a large number of cells can be infected simultaneously. Systems employing Semliki Forest Virus, Vaccinia Virus, and Retoviral vectors are used. However, drawbacks include the requirement for special precautions when engineering and preparing the viral... 

Early animal experiments have underlined the potential of vaccinia-based vector vaccines. Vaccinia virus-housing genes from HIV have clearly been found to elicit both humoral and cell-mediated immune responses in monkeys. Similar responses in other animals have been reported when surface polypeptides from a variety of additional pathogens have been expressed in recombinant vaccinia systems (Table 10.16). Human clinical trials are now in progress. 

Table 10.16. Some pathogens against which protective immunity was elicited by recombinant vaccinia vector systems. The virus invariably expressed a gene coding for a pathogen-derived surface polypeptide. The animal species in which the experiments were carried out is also listed...

Several HIV vaccine systems based upon live vectors have also been developed, in an attempt to stimulate a significant T cell as well as B cell immune response. Both envelope and core antigens have been expressed in a number of recombinant viral systems, most notably in vaccinia. The clinical efficacy of these remain to be established. 

Recombinant vaccines Vaccines that can deliver and penetrate intestinal wall via M cells Tetanus toxin, Salmonella strains, vaccinia virus vector... 

Another form of gene therapy to treat cancer involves oncolytic virotherapy. This involves the use of oncolytic vectors, which are virus-designed to home and kill the tumor cells without harming the normal cells in the body. The cancer cells are killed by cell lysis as a result of the production of cytotoxic proteins or due to the propagation of the virus itself. The viruses that have been used to produce oncolytic vectors include adenovirus, vaccinia, reovirus, HSV-1 and Newcastle disease virus. 

Puhlmann M, Brown CK, Gnant M, Huang J, et al. 2000. Vaccinia as a vector for tumor directed gene therapy Biodistribution of a thymidine kinase-deleted mutant. Cancer Gene Ther. 7 66-73. 

Hota-Mitchell, S., Clarke, M.W., Podesta, R.B. and Dekaban, C.A. (1 999) Recombinant vaccinia viruses and gene gun vectors expressing the large subunit of Schistosoma mansoni calpain used in a murine immunization-challenge model. Vaccine 1 7, 1 338-1354. 

Carroll MW, Kovacs GR (2003), Virus-based vectors for gene expression in mammalian cells Vaccinia virus, In Makrides SC (Ed.), Gene Transfer and Expression in Mammalian Cells, Elsevier Science BV, Amsterdam, pp. 125-136. 

Mackett M, Smith GL, Moss B (1982), Vaccinia virus a selectable eukaryotic cloning and expression vector, Proc. Natl Acad. Sci. USA 79 7415-7419. 

Ward GA, Stover CK, Moss B, Fuerst TR (1995), Stringent chemical and thermal regulation of recombinant gene expression by vaccinia virus vectors in mammalian cells, Proc. Natl Acad. Sci. U S A 92 6773-6777. 

This is not to say that cardiotoxicity is not seen with biopharmaceuticals. Cardiomyopathy is now a well-recognized complication of trastuzumab and and has been reported with bevacizumab treatment, in particular in combination with other cytotoxic cancer therapies [20]. Myocarditis and pericarditis are a well-documented complications of vaccinia immunization [21], and could also complicate use of a pox-virus vector for other therapeutics. In 1995 Genetics Institute suspended phase 2 cancer trials of Interleukin-12 for serious tox-icities including cardiac arrhythmia. However, such toxicities are best detected by incorporation of biomarkers for myocardial damage such as troponin-T into preclinical and early clinical studies, and continual ECG monitoring for arrhythmia in preclinical and early clinical studies, not by in vitro explorations of electrophysiology. 

Investigators are currently assessing the potential safety and efficacy of a mixed modality treatment regimen (i.e., combining a DNA vaccine with a viral vector). In this example preclinical toxicity and biodistribution studies were conducted in mice using a DNA vaccine in combination with a modified vaccinia virus Ankara based HIV vaccine to support a phase 1 trial in healthy HIV-1-uninfected volunteers [77,78],... 

In this example the DNA vaccine (pTHr-HIVA) is based on a novel direct gene transfer vector pTH [77], and the second component, MVA-HIVA, is based on a modified vaccinia virus Ankara. Both of these components contain most of the HIV-1 clade A gag protein coupled to conserved HIV-1 clade A CTL epitopes arranged in a polypeptide string that served as the immunogen [77]. 

The oncolytic viruses include adenovirus, measles, reovirus, vesicular stomatitis virus (VSV),HSV,poxvirus, and vaccinia. Specific examples include (1) ONYX-015, which is an adenoviral oncolytic virus, administered to patients with liver metastases of colorectal cancer and pancreatic cancer [29], (2) Reolysin, which is an oncolytic reovirus administered to patients with glioma [30], and (3) MV-CEA, which is an oncolytic measles virus expressing carcinoembryonic antigen, administered to patients with ovarian cancer [31]. Some oncolytic viruses are wild type and are apparently not pathogenic in humans, such as the Newcastle disease virus (NDV), which is an RNA avian paramyxovirus. PV701, a naturally attenuated, replication-competent strain of NDV, has been administered to patients with advanced solid tumors [32], The applicability of oncolytic viruses as a therapy for clinical oncology trials is due to their potential selectivity the ability to kill tumor cells but not normal cells. However, the level of attenuation of viral replication in normal cells is limited for most oncolytic vectors. 

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