2.2 RETROVIRAL VECTOR PACKAGING CELL LINES 
Retroviruses are positive-strand RNA viruses that pass through a double-stranded DNA stage after infection (3132). 
Viral DNA integrates into the host genome to form a provirus, which contains the viral genes encompassed on either 
end by viral long terminal repeat (LTR) elements which are required for integration. After integration, viral genes are 
expressed from the provirus by utilizing host cell machinery and transcriptional promoter and termination cis 
elements found within the viral LTRs. The full length viral RNA transcript (the next generation viral genome) is 
recognized via a specific "packaging signal" (HO found in the viral RNA genome and encapsidated into an infectious 
viral particle by the viral proteins. The retroviral life cycle is generally non-lytic for the cell, and allows continuous 
viral jM’oduction over a long period of time. 
The retroviral vector used in these studies is based upon Moloney murine leukemia virus (MoMLV), that was 
engineered so that its extraneous viral coding sequences have been replaced with a desired gene (32-34), such as y- 
DFN. Unlike true viruses, the recombinant vector is incapable of replication since its genome lacks all of the vir^ 
structural genes. Any gene of interest can replace the protein coding region of the viral genome and become 
packaged within a packaging cell line (PCX) into virion particles for transduction, provided that it is contiguous with 
the essential cis elements (i.e., T, LTRs, and sequences required for priming of DNA synthesis) of the genome. 
The viral proteins needed for enc^sidation and vector production must then be supplied in a PCL. The PCX is a 
cell line which expresses the MoMLV structural proteins, and as a result produces empty virion particles which lack 
the viral genome required fw replication. Viral provector DNA introduced into the PCL will act as a template for the 
production of the RNA vector genome, which is then packaged into the previously empty virions, generating 
retroviral vectw particles (3334). 
For clinical studies, the most important safety concern with the use of retroviral vectors is the inherent propensity of 
murine retroviral PCLs to genaate replication-competent retrovirus (RCRV) after introduction of a vector (33-36). 
This can theoretically occur by homologous recombination between the retroviral provector DNA and either 1) the 
DNA encoding the MoMLV structural genes present in the PCL (36), or 2) the endogenous proviruses found 
universally in murine cells (37), the species from which all the MLV-based PCLs previously used for human gene 
therapy studies w^e genwated. In facL even murine cell lines lacking vector can he induced to produce xenotropic 
RCRV (38) which can replicate in human cells. 
Another safety concern with murine PCLs used for retroviral vector production is that endogenous defective 
proviruses in the murine genome are expressed (e.g., VL30 sequences), recognized by the retroviral structural gene 
products of murine PCLs, and delivered and exj^essed in target cells with an efficiency at least comparable to that of 
the desired vector (39). The delivery of these undesirable retroviral-like sequences and the possibilities for generating 
replication-competent retrovirus suggest that murine cell lines may be less than ideal for the production of murine 
retroviral vectors for human thaapeutics. 
Due to the safety risks associated with the generation of RCRV by homologous recombination with endogenous 
sequences, we have developed MLV-based retroviral PCLs from a canine cell line, D17. The MLV structural genes 
("gag/pol" and "env") are also genetically unlinked, to decrease the chance of retrovirus generated by homologous 
recombinations between the therapeutic pro-vector DNA and the DNA encoding the MoMLV structural genes present 
in the PCL. These changes should help alleviate many of the safety concerns described above. These vectors have 
been previously used in several RAC approved gene therapy protocols. 
2.3 CLINICAL APPROACH 
We propose to utilize retroviral vectors to deliver and express the y-EFN gene to human tumors in order to improve 
active immunity for human malignancies. It is hoped that this approach will improve antigenic presentation and 
subsequent tumor-specific immune activation in cancer patients by creating an optimal microenvironment around the 
tumor cells for the activation of cell mediated immune responses. Short-term tumor cell cultures will be established 
from human tumor biopsies and/or bone marrow metastases. The resultant cell cultures will be transduced with the 
y-IFN retroviral vector. After G418 selection, lethal irradiation, and quality control testing, the lymphokine- 
expressing autologous tumor cells will be injected back into the patient An increase in specific immunity against 
the gene-modified tumor cells may result in the clearance of the endogenous unmodified metastatic tumors (see Fig 1 
for a diagrammatic summary of the approach). 
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Recombinant DNA Research, Volume 19 
