HEPATOCELLULAR TRANSPLANTATION AND TARGETING GENETIC MARKERS TO HEPATIC CELLS 
I 
technological basis for gene therapy in detail (Ledley, 1989) (Appendix G) while another L 
surveys the issues involved in restoring metabolic homeostasis in inborn errors of ■ 
metabolism by gene transfer (Ledley, 1990) (APPENDIX G) . The last review explores | 
practical issues in the application of gene therapy technology to clinical research 
(Ledley, 1991) (Appendix G) . ii 
I 
The major focus of research towards gene therapy has been to develop methods for ! 
efficient gene transfer into somatic cells. There is a general consensus that 
retroviral vectors based on Moloney murine leukemia virus (MMLV) , such as the 
amphotrophic vector LNL6, are presently the vectors of choice for experiments in somatic 
gene therapy. Retroviral vectors are constructed essentially according to the scheme ^ 
developed by Mann et al (1983) . This construction involves producing a defective 
retroviral genome by recombining a gene fragment (generally a cDNA) with the LTR and i 
elements from MMLV. The RNA transcript from this clone is then packaged into a viral j 
particle produced by another recombinant viral gene which encodes synthesis of a viral 
particle, but lacks the ^ or LTR elements required for packaging (figure 1). This ^ 
technique produces a "defective retrovirus" which is capable of transducing a single 
cell but does not encode viral proteins and is incapable of propagating viral 
proliferation. 
This methodology has improved with: i) development of improved packaging cell lines 
(including PA317, Miller and Buttimore, 1986) which are not liable to recombination 
between the incomplete retroviral constructs (Markowitz, 1988a; 1988b; Danos and | 
Mulligan, 1988) ; ii) the recognition of extended ^ sequences which improve the ]t 
efficiency of retroviral production (Armentano, 1987; Adam and Miller, 1988; Bender et ! 
al, 1987); and iii) with additional mutagenesis to eliminate potential retroviral i 
reading frames or other sequences which could produce novel gene products (Bender et al, |: 
1987, Miller and Rosman, 1989). Retroviral vectors have many features which are helpful jj 
for gene transfer: the ability to construct defective retroviruses which do not n 
contain genes for any viral proteins; 11) the wide host and tissue trophism; 111) the | 
high efficiency of stable integration following infection; Iv) the lack of 
rearrangements or deletions surrounding the site of insertion into the genome; and v). |*‘ 
the ease of constructing recombinant with promoter, enhancer, and coding sequences j. 
within the retroviral framework (reviewed Ledley, 1989). I 
i: 
Concerns have been expressed about the safety of retroviral vectors (reviewed p' 
Ledley, 1989), particularly: 11 the risk of recombination with endogenous viruses to 
form novel infectious agents; ii) the risk of insertional mutagenesis of an essential 
genetic element; or iii) activation of a proto -oncogene by insertion of retroviral 
promoter elements in the vicinity of a proto - oncogene , iv) inadvertent packaging of a L 
wild type viral genome by packaging cells during production of the defective retrovirus 
(Scadden et al, 1990). It should be noted that the vectors which we propose to use in 
this study has been expressly designed to avoid many potential complications, and the j 
theoretical risk of complications is calculably small. ^ 
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Recombinant DNA Research, Volume 14 
