Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

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Chapter | 2 Comparative Medical Genetics
cats as an intermediate between the murine models and
applications in humans. Hence, gene therapy, though promising
for humans and animals alike and potentially simple
to administer, is still an experimental approach for inborn
errors of metabolism .
There are several ways to introduce a new gene into a cell.
One of the more common current approaches uses viruses that
act as vectors to target and transport DNA into a cell, often
into the nuclear DNA. The viral vectors are modified to make
them replication incompetent and contain additional genetic
material, usually including at least a promoter in addition to
the cDNA of the gene of interest. Many viruses have been
used for gene transfer, including recombinant herpesviruses,
lentiviruses, adeno-associated viruses (AAV), adenoviruses
(Ad), and Sendai virus (reviewed in Verma and Weitzman,
2005 ). Although gene therapy is a promising technique for
therapy for the LSDs, concerns remain, particularly those
involving carcinogenesis. Integration can interfere with the
normal function of nearby genes ( Hacein-Bey-Abina et al. ,
2003 ; Schmidt et al. , 2005 ) with ensuing deleterious effects.
Thus, although recombinant retroviral vector gene therapy
cured 80% of X-linked Severe-Combined Immunodeficiency
(SCID) patients, the development of leukemia in some
patients made real what had been a hypothetical risk since
the early experiments in the 1990s. Also, the development of
tumors in aged Mucopolysaccaridosis (MPS) VII mice that
received recombinant adeno-associated viral vectors at birth
have been reported ( Donsante et al. , 2001 ). Other limitations
have included an immune response to the vector or the
transgenes they encode, particularly when individuals have a
null mutation resulting in no protein production. In general,
viral vectors are made replication incompetent by various
techniques, such as removing genes encoding structural viral
genes, limiting concerns about shedding vector for extended
periods, and reducing the potential immune responses against
the virus. However, readministration of a vector has a significant
potential to induce an immune response. The search for
new vectors and ways to modify existing vectors to reduce
these limitations is ongoing. Three viral vectors currently in
common use include the following:
1. Retroviruses are RNA viruses that reverse transcribe
their single-stranded genome and can integrate into host
chromosomes ( Fields and Knipe, 1986 ). Some murine retrovirus
vectors—for example, those based on murine leukemia
viruses—transduce only dividing cells. Lentiviruses
can also transduce nonreplicating cells ( Fields and Knipe,
1986 ) and have been generated from the human immunodeficiency
virus and feline immunodeficiency virus ( Johnston
et al. , 1999 ; Stein and Davidson, 2002 ) among others. The
tropism of retroviruses can be modified by providing an
alternative envelope glycoprotein during virus production,
leading to broader cell-type targeting and enhanced stability
upon enrichment ( Stein et al. , 2005 ; Wong et al. , 2004 ).
2. Adenoviruses are nonenveloped double-stranded
DNA viruses ( Fields and Knipe, 1986 ), which can infect a
variety of both quiescent and proliferating cells. The vectors
have space for large transgenes; however, transgene expression
is often transient because of host immune responses to
the vector ( Stein et al. , 1998 ; Yang et al. , 1996 ).
3. Adeno-associated viruses (AAV) are small singlestranded
DNA viruses that do not produce disease but are,
as the name implies, associated with adenoviruses ( Fields
and Knipe, 1986 ). This relationship renders AAV’s replication
incompetent without a helper virus, usually an
adenovirus or a herpesvirus. Many serotypes have been
developed ( Chiorini et al. , 1997 ; Schmidt et al. , 2004 ),
with most showing distinct tissue tropism ( Gao et al. ,
2005 ).
1 . Approaches for Gene Therapy Using
Lysosomal Storage Diseases as Examples
a . Ex Vivo Gene Therapy
The usual strategy in this approach is to modify autologous
patient cells in vitro , returning them to the patient to create
an “ enzyme factory ” to secrete a specific enzyme into the
extacellular fluid to be taken up by other cells elsewhere
in the body, thereby “ cross-correcting ” them. The most
common therapeutic target is the pluripotent hematopoietic
stem cell. Not only can these cells secrete enzyme into the
circulation, they can repopulate fixed macrophages in liver,
lung, brain, and elsewhere allowing widely distributed sites
of local enzyme production. The rationale for this approach
has been from the experience of heterologous bone marrow
transplantation in animals and children ( Birkenmeier et al. ,
1991 ; Breider et al. , 1989 ; Consiglio et al. , 2004 ; Follenzi
et al. , 2002 ; Hoogerbrugge et al. , 1988 ; Miranda et al. ,
1998 ; Sands et al. , 1993 ; Taylor et al. , 1986 ; Walkley
et al. , 1994 ; Yeager et al. , 1984 ). Gene therapy of autologous
cells avoids the difficulties of finding a histocompatible
bone marrow donor, and if engineered to have very
high gene expression, the transduced cells will deliver
substantially larger amounts of enzyme than normal cells
( Biffi et al. , 2004 ).
Ex vivo transduction and transplantation into enzymedeficient
mouse models of LSDs has shown efficacy in
vivo using murine retroviruses ( Miranda et al. , 2000 ; Wolfe
et al. , 1992 ). Thus, hematopoietic-directed gene therapy
may be effective for the treatment of the systemic disease
associated with a number of LSDs. However, thus far, this
approach has not achieved the success in clinical trials that
was seen in the animal models ( Dunbar et al. , 1998 ), possibly
because of a lack of conditioning to reduce resident
bone marrow. Lentiviral vectors may be a better choice
over murine retroviruses, because they transduce nondividing
cells and show persistent in vivo expression.