Applications of Gene Therapy - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 299

The human enzyme was removed by a mouse monoclonal antibody that did not react
with mouse glucocerebrosidase protein. When GC-encoded viral mRNA from the
mouse cells was packaged into retroviral particles, it was possible to insert the gene
into fibroblasts and lymphoblasts from patients with GD. The cells were subjected
to selective pressure with G418, and the surviving fibroblasts and lymphoblasts were
found to have normal amounts of GC activity [298] . In another study, primary murine
fibroblasts were transduced with retroviral vector having GC cDNA. Further, these
transduced myoblasts were implanted into syngeneic C3H/HeJ mice. The results dem-
onstrated that transplanted mice sustained long-term expression of transferred human
GC gene in vivo , up to 300 days. Human GC in the circulation reached levels of
20-280 units/mL of plasma. Immunohistochemical studies of the target organs revealed
that the secreted human GC is taken up by macrophages in the liver and BM [299] . In
another investigation, recombinant adeno-associated viral (r-AAV) vectors containing
human GC cDNA were used to express human Gaucher fibroblasts. This r-AAV vector
mediated efficient expression of human GC in human Gaucher fibroblasts. GC activi-
ties were increased both in normal fibroblast and in Gaucher fibroblasts, for a long
period. Upon intravenous administration of this system via the hepatic portal vein and
tail vein of wild-type mice, significantly high levels of GC activity were observed in
different organs. Therefore, r-AAV vector-mediated gene transfer may provide a thera-
peutic approach for the treatment of GD [300] .
Gaucher disease can be cured by replacing the patient's defective hematopoietic
stem cells with genetically normal stem cells. There has been success in transducing
murine and human BM progenitors with retroviral, lentiviral, and adeno-associated
viral vectors containing DNA encoding human GC. In a study, retroviral transduc-
tion of autologous BM, followed by transplantation in mice, showed that transduced
transplanted BM repopulates macrophages and central nervous system microglia,
and GC cells begin to infiltrate the liver, lung, brain, and spinal cord by 3 months
after transplant. In the brain, 20% of the total microglia was replaced with donor
cells expressing GC by 3 to 4 months after transplant [301] . Another study, involving
transplanting BM stem cells transduced with a retroviral vector containing the human
GC cDNA into mice resulted in successful engraftment, long-term expression of the
transgene, and safety of multiple transplants in recipient mice [302] . Other studies
showed that transplantation of bone-marrow-derived cells transformed with a lenti-
virus encoding for GC results in long-term expression of the human GC gene in vivo
[303] . Further, AAV8-mediated expression of GC ameliorated the storage pathol-
ogy in the visceral organs of a mouse model of GD [304] . A study showed that both
transplantation of BM and gene therapy through retroviral transduction of BM from
GD mice prevented development of disease and corrected already established GD
phenotype-viable mice with characteristic clinical symptoms of type 1 GD [305] .
6.7.2 Human Clinical Trials
Retroviral gene transfer of the GC gene to hematopoietic progenitor and stem cells
showed promising results in animal models and corrected the enzyme deficiency in
cells from Gaucher patients in vitro . A clinical trial was initiated to check the safety