recent studies suggest that the problem of expression of transferred genes is being overcome 
in a variety of systems' 59 ' 65 ' 7478 ' 
Clinical studies of gene therapy are increasing rapidly. Evaluation of the safety 
and effectiveness of the approach are the subject of intense investigation 1791 . 
IV. PRE-CLINICAL STUDIES 
A. DEFINITION OF THE TARGET CELL AND EFFICACY OF THE GENE PRODUCT 
As the first step toward this goal, we cloned the gene for glucocerebrosidase' 13,151 . 
We demonstrated that the cDNA in a retroviral vector, pWE-GC, provided by Dr. Richard 
Mulligan, could correct the deficiency of GC in fibroblasts from a patient with Gaucher 
disease' 86 ' 81 '. Earlier we had demonstrated that allogeneic bone marrow transplantation 
resulted in the reversal of the disease process 123 ’. These results supported the hypothesis 
that gene therapy might be used to treat the disease by transfer of the GC gene to bone 
marrow cells. In addition, we demonstrated that targeting GC enzyme to macrophages resulted 
in a clinical response in a patient with Gaucher di sease' 9,26,271 . This result supports the 
hypothesis that the gene product is therapeutic. These experimental approaches also 
provided some encouragement that reconstitution of only a part of the normal enzyme capacity 
of a tissue would be sufficient to treat the disease. We concluded from these studies that 
gene therapy was possible as a treatment for Gaucher disease, provided that a fraction of 
tissue macrophages could be transduced with and would express the GC gene. 
B. VECTOR CONSTRUCTIONS 
The gene therapy approach to Gaucher disease required a vector that would result in 
efficient transduction and expression in hemopoietic cells. Dr. Donald Kohn joined the 
group to work on this project. He constructed a set of retroviral vectors derived from the 
Moloney murine leukemia virus (MMLV) placing the GC cDNA under the control of either its 
own promoter or one of several strong heterologous promoters. These N2 derived vectors were 
replication defective and were raised in the ecotropic virus producer cell lines to titers 
of 10 5 to 10 7 . Viral supernatants were used to infect 3T3 cells to test the vectors for 
efficiency of infection and expression of the GC gene. In 3T3 cell targets, transduction 
efficiency for all the vectors was essentially the same and approached 100%. The vector 
giving the most consistent results in terms of expression of the GC gene was a construct 
called N2-SV-GC. This vector resulted in about a two fold increase above control in human 
GC RNA transcripts in Northern blots or in GC protein on Western blots using a human 
specific monoclonal antibody (Mab-8E4), or in activity of glucocerebrosidase in extracts. 
This expression surpassed that obtained from vectors using either homologous GC gene 
promoter regions or the HSV-TK promoter 182 '. N2-SV-GC was used to study the transfer of the 
human gene to cells growing in long term bone marrow cultures (LTBMC)' 41 ’. The goal of these 
experiments was threefold; first, to obtain a supply of transduced stem cells from LTBMC 
J which could be used to study GC gene transfer in mice with eventual application to the GD 
| mouse model. The second purpose was to study methods to support and expand cycling stem 
! cells in culture as a source of marrow repopulating cells that might be adapted later for 
i human applications. Third, the plan was to demonstrate that transduction of human Gaucher 
bone marrow by a normal GC gene would correct the enzyme deficiency in HSC marrow cells and 
their progeny. 
I 
The results of studies of N2-SV-GC in LTBMC demonstrated that N2-SV-GC was capable 
of infecting LTBMC. The transduction efficiency was high and expression of the GC gene 
could be detected in some spleen 'foci' in mice transplanted with bone marrow infected by 
N2-SV-GC. The amount of expression approached that seen in normal fibroblasts. These 
experiments demonstrated that the first goal could be partially met i.e. that LTBMC could 
be transduced with the human GC gene. 
! 
Recombinant DNA Research, Volume 17 
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