Biomedical Engineering Reference
In-Depth Information
cells and does not integrate into the genome. The carrying capacity is attractive for the
insertion of large transgenes or complex gene cassettes. However, the safety of HSVs is
unknown.
12.1.3.4 Adenovirus
Adenovirus is easy to produce in high titers (10
10
-10
12
pfu/ml), can infect non-dividing cells
and has a capacity of 7-8 kb. It is transient because it does not integrate into the genome.
It has been a preferred vector for numerous experiments, but it was a disastrous failure in a
human trial. The adenovirus is not safe because it produces proteins that cause a graft-host
reaction. The immune and inflammatory responses to adenovirus and the proteins it generates
was predictable and its use in humans was not only tragic for the loss of a subject's life
(17-year-old Jeremy Gelsinger), but also a serious setback to other gene therapies as a novel
therapeutic approach.
12.1.3.5 Adeno-associated virus
AAV is not related to adenovirus, despite the name. AAV is a human parvovirus that can
infect non-dividing cells. It is actually very common in humans and apparently harmless.
AAV is long lasting and stable. The wild type integrates into chromosome 19. Recombinant
adeno-associated virus (rAAV) also integrates [25], although each new rAAV needs to be
tested for which chromosome it integrates into. When stripped of its gag and pol genes
down to the bare inverted terminal repeats, an AAV vector appears to be non-toxic and does
not induce host immune responses because it does not produce foreign proteins. Uptake
efficiency depends on multiple of infection (MOI, i.e. number of viruses, or DNA particles,
per cell) (see Figure 12.1).The main disadvantage for AAV is its low carrying capacity
(4.7 kb) .But it can be produced in very high titers (more than 10
12
pfu/ml). It is being used
in clinical trials and has several advantages for gene modification and cell transplantation.
Recently, the serotype of AAV has become recognized as an important consideration in gene
transfer into particular tissue. Cheng
et al
. [26] have shown that serotype 8 is more efficient
for transfecting the pancreas than the standard serotype 2. Others have shown preferential
tissue transfection for serotype 5 in the liver [27] and lung [28].
12.1.3.6 Reporter genes
To label cells with an internal marker so that the cells can be identified after transfection
with AAV, a reporter gene can be inserted into the AAV. To label cells with an internal
marker so that cells transfected with AAV can report activity of a viable cell, fluorescent
genes [29-31] can be inserted into the AAV; for example, intracellular green fluorescent
protein (GFP) delivered by AAV, as shown in Figure 12.1. Similar effects are seen with
Luciferase (Luc) or
β
-galactosidase (Lac Z). The gene sequence of these reporters can be
inserted into any of the vectors described above and mark the uptake and expression of
invisible proteins expressed by the transgene delivered by the vector. Each cell marker
has its own advantage or disadvantage. An advantage of GFP is that it is visible using a
highly sensitive fluoroscope so that the cells can be located, even under the skin in tissues
and tumors. Luciferase has the advantage that it is quantifiable using luminometers, dual
luciferase assays or relative luciferase gene expression. Lac Z is quantifiable but not visible
in vivo
. At the next level of sophistication, a cell- or tissue-specific promoter is spliced with
