V. 
EXPERIMENTAL DESIGN 
A. OVERVIEW 
The hypothesis of this study is that autologous bone marrow transplantation of 
genetically corrected peripheral blood stem cells (PBSC) will result in a sustained reversal 
of the phenotype in patients with Gaucher disease. The specific aims to be achieved are: 
1) Transfer of the human GC gene into PBSC (CD34 + ) obtained from patients with Gaucher 
disease. 
2) Transplantation of transduced PBSC autologously to patients. 
3) Estimation of engraftment of CD34 + cells by measurement of the carriage and 
expression of the transferred gene and its duration in peripheral blood leukocytes 
(PBL). 
4) Assessment of the clinical effects of transplanting genetically corrected PBSC in 
patients with Gaucher disease. 
This study is a clinical trial of gene therapy for patients with Gaucher disease. 
It involves the use of G-CSF to stimulate the release of CD34 + cells into the blood from 
the bone marrow. These cells are harvested by leukopheresis, density gradient 
centrifugation, and percolation over an immunoaffini ty resin (CEPRATE^SC) to harvest CD34 + 
cells. The cell fraction contains 50-80% CD34 + cells and is enriched for cells capable of 
long-term reconstitution of the bone marrow (stem cells). 
Peripheral blood CD34 + cells enriched for stem cells (PBSC) will be transduced with 
the normal GC gene by exposure to supernatants from the amphotrophic producer lines of the 
vector once a day for four days. The PBSC will be collected and washed. An aliquot of the 
harvest will be immediately lysed and assayed for gl ucocerebrosidase activity. Only 
harvests with GC activities within the range of normal will be used in transplantation 
studies. Cells also will be assessed for carriage of the GC gene by Southern analysis and 
will be tested for contaminating materials. Cells will be viably frozen until the results 
of tests are complete. 
After obtaining negative results in a routine sterility test and negative tests for 
helper virus in the supernatant of the ex vivo transduction mixture, patients with Gaucher 
disease will be transplanted with 2 x 1 0 6 /kg PBSC. This is the dose that has become 
standard as a reconstituting dosage of PBSC. In this study we will evaluate whether 
patients will require myeloabl ation to achieve a successful result. The decision to use 
myeloablative preparation will be made on the basis of correction of enzyme deficiency in 
the leukocytes of patients transplanted with genetically corrected PBSC who have not 
received myeloablative preparation. Results in the first two patients studied will be used 
to make this decision. Patients usually engraft rapidly with PBSC and could be considered 
to be complete within one month. Therefore, assay of PBL for GC activity will be performed 
after that interval of time. The assays of enzyme activity will be used to make the 
decision. The results in these first two patients will determine the approach to be used 
in all subsequent patients studied. If the amount of GC in leukocytes is not increased 2 
fold above the deficient level, it will be concluded that an inadequate number of corrected 
stem cells have engrafted. If this is the case, patients will be prepared with 
cyclophosphamide (8g/m 2 ) to partially ablate the genetically defective marrow. This 
preparation should permit replacement of at least part of the bone marrow with genetically 
corrected cells. The approach of partial marrow ablation has been chosen to reduce the 
risks to the patient. 
After having established the restoration of enzymatic activity and carriage of the 
transferred gene, patients will be studied for the clinical responses to the therapy. This 
includes repeated measurements of clinical and laboratory parameters known to be abnormal 
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