Applications of Gene Therapy - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 300

and feasibility of retroviral transduction of peripheral blood (PB) or BM CD34 cells.
The CD34-enriched PB cells or CD34-enriched steady-state BM cells were mobilized
using G-CSF and transduced ex vivo with retrovirus expressing the human glucocer-
ebrosidase cDNA. Patients 1 and 2 were given transduced PB cells, while patient 3
was given transduced BM cells by infusion. When gene marking was demonstrated in
patients, patient 2 had vector-positive PB granulocytes and mononuclear BM cells at
1 month postinfusion and positive PB mononuclear cells at 2 and 3 months postinfu-
sion. Patient 3 had a positive BM sample at 1 month postinfusion but not afterward
[306] . A second trial conducted also involved three patients. The G-CSF-mobilized
CD34 cells were transduced in a long-term culture, with a transduction efficiency
of 0.01-0.1%. An immediate twofold increase in enzyme level was detected, but there
was no engraftment of transduced cells [307] . In a third clinical trial, transplants of
genetically corrected, autologous CD34 cells were performed. All of the subjects
were able to mobilize CD34 cells using G-CSF. Transduction efficiency averaged to
20% and was improved consistently with adjustments to the protocol. The activity of
glucocerebrosidase increases above the deficient levels and returns the activity of GC
to normal levels. Total PB leukocytes and CD34 cells prepared from the blood of
study subjects were positive by PCR for the GC transgene [308] .
6.8 Applications of Gene Therapy in Hemophilia
Blood coagulation is an important part of the body's homeostasis mechanism. Upon
injury to the blood vessel, the coagulation cascade of the body becomes active via
extrinsic and intrinsic pathways. Factors VIII (FVIII) and IX (FIX) are an important
part of the intrinsic pathway of coagulation cascade. A deficiency or dysfunction of
either FVIII or FIX leads to bleeding following vascular injury. Hemophilia is an
X-linked inherited bleeding disorder characterized by severe uncontrolled hemor-
rhagic episodes that can even be fatal. Hemophilia A and B resulted from a subnor-
mal level or deficiency of essential cofactors, FVIII and FIX, respectively. The defect
in FVIII and FIX synthesis occurs due to mutation in the genes synthesizing them,
respectively [309] .
Currently, hemophilia is treated with protein replacement therapy using either
plasma-derived or recombinant coagulation factors. The replacement therapy is
extremely effective, but some patients develop antibodies after replacement therapy.
Hemophilia is one of the targets for gene therapy for the following reasons:
1. FVIII and FIX, which are responsible for hemophilia A and B, are well characterized and
can be monitored easily in the patient's blood.
2. A number of hemophilic animal models (mice, canines, rabbits) are available to test gene
therapy protocols.
3. Hemophiliacs are good candidates to determine side effects of gene therapy protocols due
to their long life expectancy.
4. Gene therapy may possibly result in the reduction of treatment costs over replacement
therapy.