Applications of Gene Therapy - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 301

6.8.1 Preclinical Studies
Both ex vivo and in vivo strategies have been used for the preclinical trials of hemo-
philia in experimental models [310] . For hemophilia A, most of the studies involved
transfer of FVIII cDNA either as such or with the deletion of B-domain-deleted cDNA-
using retrovirus. The removal of B-domain significantly reduces the size of FVIII
cDNA, without affecting the function of the protein (the complete and the B-domain-
deleted FVIII variants are virtually identical in functional assays) [311] . A number of
cell types have been used for retrovius-mediated expression of FVIII gene-like fibro-
blasts [312] . The retrovirally transduced fibroblast cells secreting FVIII of human ori-
gin were implanted in nude mice. The cells isolated from the graft after 8 weeks had
the capacity to secrete FVIII, when regrown in culture [313] . Apart from fibroblasts,
other cell types have also been used for gene therapy of hemophilia, using retrovirus-
mediated ex vivo transduction. These include endothelial cells [314] , myoblasts [315] ,
and hematopoietic progenitor cells [316] . Conclusively, these studies demonstrated that
modifications of hepatic cells are not mandatory for the expression of FVIII.
The in vivo strategy was designed for liver-directed gene therapy for hemophilia A,
which needs the proliferation of hepatocytes. The rapid proliferating capacity of
hepatocytes in newborn mice has been utilized in a study resulting in high-level
expression of FVIII [317] . The transposition of plasmid-expressing FVIII into the
host chromosome is another approach to treat hemophilia. For example, the polyeth-
ylenimine and sleeping beauty transposition system was used for FVIII expression
in mice models [318] . The immune system of the host provides barriers for FVIII
expression. Therefore, strategies have been designed to overcome this problem, for
example, the use of newborn mice with immature immune systems [319] .
Like hemophilia A, the preclinical studies using the ex vivo approach were car-
ried out for hemophilia B. A major advantage of hemophilia B in the development
of gene therapy strategies is the relatively small size of FIX cDNA (~1.4 kb of cod-
ing sequence) that could be easily inserted into many different gene transfer vectors.
When transduced with FIX cDNA, a variety of cultured cells secreted functional FIX
[320] . The various cell types studied for FIX transduction are fibroblasts, myoblasts
[321] , keratinocytes [322] , and hematopoietic cells [323] . However, transplantation
of the transduced fibroblasts into mice resulted in transient FIX plasma levels that
were lower than would be expected on the basis of the FIX secretion in vitro [324] .
FIX were also efficiently delivered in large animal models like hemophilic dogs
using an in vivo approach either alone or with HGF, which improved the expression
of FIX [325,326] . Adenovirus and adeno-associated virus vectors have also been uti-
lized for effective delivery of FIX [327,328] .
6.8.2 Human Clinical Trials
The success of preclinical studies in animal models led to the development of human
clinical trials. Both viral and nonviral delivery methods have been utilized so far, using
both ex vivo and in vivo approaches. In a clinical trial, the nonviral method was used
for the delivery of plasmid containing FVIII cDNA in patients with severe hemophilia,