Gene Therapy for Parkinson’s Disease - Viral Vector Approaches in Neurobiology and Brain Diseases - page 181

way to determine if the gene of the promoter candidate is
expressed in the animal model is to perform a PCR.
4. It is recommended to use the Gateway system (Invitrogen) for
cloning since the transgene can be easily changed, to for exam-
ple a therapeutic gene, if required.
5. Some promoters show a different specifi city depending on the
site of injection. One promoter showing this pattern is the
cytomegalovirus (CMV) promoter. Transgene expression
from lentiviral vectors containing the CMV promoter is mainly
restricted to neurons in the striatum, but if the vector is
injected into the midbrain the portion of glial cells increases
[ 39 ]. The promoter candidates should therefore be evaluated
in the brain nuclei where it is going to be used.
6. It is recommended to inject a small amount of vector (1-2 l for

striatum for example) in the evaluation experiments. This is to
avoid having too many cells expressing the transgene since this
will complicate the analysis of cell specifi city and effi ciency.

μ

7. It is recommended to use a glass capillary mounted on a
Hamilton syringe during the injections. This decreases the tis-
sue damage and increases the precision of the injection.
8. Additional staining to determine the level of dopamine deple-
tion and infl ammation should be performed. Differences in
these parameters may alter the specifi city and effi ciency of the
vector.
9. Even though there are several studies identifying tissue-specifi c
miRNA, the miRNA specifi city for a particular application will
need to be validated by the researcher.
10. The promoter needs be chosen carefully when using miRNA
detargeting. A very active promoter can lead to high transcrip-
tion of messenger RNA that can potentially saturate the
miRNA and stop its function, becoming a miRNA sponge
[ 40 ]. Therefore, avoid promoters that lead to a very high con-
stitutive activity.

5 Conclusion

Patient microarray data provides an opportunity to fi nd promoters,
relevant for the disease, for use PD gene therapy paradigms.
Detargeting using miRNA target sites may then be used to ensure
cell specifi city. By using a vector that is both cell specifi c and rele-
vant for PD, gene therapy could potentially be made safer and
more benefi cial for the patient.