Esra Comert-Morishige

Bio 485: Final Paper

March 9, 2015
Phenylketonuria: Targeting Muscle Tissue to Treat a Metabolic Disorder
Imagine you have a two-year-old child. Its around 2pm and you try again,
fruitlessly, to feed her. Shes been refusing her formula all day because this formula is all
shes ever been allowed to eat and she is not having it today. Imagine you have few other
food options to try because any foods containing the amino acid Phenylalanine, if
ingested by her, can cause horrifying symptoms such as mental retardation, skin and hair
disorders, reduced growth, and neural malfunctions that can cause tremors and seizures.
If she continues to ingest Phenylalanine, she can develop serious behavioral disorders
such as anxiety and hyperactivity1.
Your child has a rare genetic disorder called Phenylketonuria, for which all infants
born in the US are screened for at birth. Her body doesnt produce an enzyme called
phenylalanine hydroxylase, which an enzyme that is responsible for breaking down
phenylalanine in the body into usable components, thus preventing the toxic buildup of
this amino acid. Phenylalanine is present in many protein-rich foods and is an essential
element of the human diet. However, because people with Phenylketonuria cant break
down excess phenylalanine, this amino acid builds up in the blood stream. Traditionally,
the long-term treatment plan for people with Phenylketonuria is sustained dietary
restriction of phenylalanine-containing foods, supplemented with formulas containing
vitamins and minerals and all essential amino acids except phenylalanine2. However,
there have been several nutrition imbalances reported following the use of these
supplements and often, children become non-compliant with the regular ingestion of

formula3. Though the disease can impact several different tissues (since excess
phenylalanine is circulated through the blood), the brain is particularly sensitive to this
amino acid. Thus, brain damage is commonly associated with untreated
Phenylketonuria, making the need for effective treatment plans all the more urgent.
Aside from nutritional therapy, a few other treatment options have been
researched. These include the use of Large Neutral Amino Acid (LNAA). LNAA is
similar in shape to phenylalanine and can therefore compete with it for access to cellular
transporters. The use of LNAA can thus reduce the amount of phenylalanine that can
cross the gastrointestinal and blood brain barrier, reducing the amount of phenylalanine
absorbed by the brain4. Alternatively, a molecule called tetrahydropterin (BH4) has been
shown to reduce phenylalanine concentrations in the blood of people with mild
phenylketonuria by promoting the stability of phenylalanine hydroxylase5. However,
these approaches attempt to treat the disease at a peripheral level by targeting how
phenylalanine or phenylalanine hydroxylase behaves in the body. Furthermore, they have
shown the most success in people with mild forms of the disease. In comparison, I am
proposing a therapy mechanism using gene-correction to target the mutated PAH gene in
patients. If successful, this therapy would provide a cure for people with mild to severe
forms of Phenylketonuria.
The gene implicated in Phenylketonuria is called PAH. This gene provides
instructions to make phenylalanine hydroxylase under normal conditions. There are
varying types of PAH mutations that lead to different levels of loss-of-function for this
gene. Milder forms of PKU, such as variant PKU and non-PKU hyperphenylalaninemia,
are associated with less severe PAH mutations. The gene is located on chromosome 12

and the most common mutations change only single amino acids in the PAH sequence.
Other mutations delete small portions of the gene or interfere with the genes ability to
be translated into phenylalanine hydroxylase. An example of a common mutation is the
replacement of arginine with tryptophan at position 408. The disease is inherited with an
offspring receives two mutant alleles (one from each parent). Because of the recessive
nature of the mutant allele, parents often dont know that they are carriers (heterozygous
individuals dont show any PKU symptoms)6.
Gene therapy research surrounding Phenylketonuria is currently underway. Many
researchers have focused on the liver as a potential source of stem cells for gene therapy.
The liver is a major production site for phenylalanine hydroxylase in healthy individuals,
so this organ is a reasonable target for the correction of the PAH gene. Though this gene
has been successfully corrected in mice through the use of an adenovirus (containing the
corrected PAH gene) targeting hepatocytes, the results of such research have been shortlived. The vectors genome has not been shown to successfully integrate into the mouse
hepatocytes DNA, so the corrected gene (and its function) is lost upon hepatocyte
regeneration. The secretion of phenylalanine hydroxylase by the liver is minimal and
short-lived, since hepatocyte regeneration occurs so frequently. Furthermore, the
reinjection of the same adenovirus has been shown unsuccessful because of an immune
response that destroys the vector 7.
I propose two modifications to this gene therapy approach that might help
ameliorate the issues with the original treatment plan. Firstly, there is research that
supports the hypothesis that using different types of adenovirus vectors can prevent their
antibody-mediated destruction in the body in trials where multiple injections are

necessary8,9. Secondly, I propose that we find an alternative target cell population for
viral vectors, since hepatocytes have proved ineffective in their ability to uptake and
maintain corrected genes in their DNA. New research has proposed the intramuscular
injection of AAV2 pseudotype 1 (rAAV2/1) or rAAV2/8 vectors (containing genetic
information for correct phenylalanine hydroxylase and tetrahydrobiopterin production) in
mice as a potential treatment option for phenylalanine hydroxylase deficiency9. This
method has proven more successful in terms of long-term gene retention than previous
studies targeting hepatocytes.
Concentrating on muscle tissue is less invasive, less expensive, and more effective
than targeting the liver directly. This research has shown that PAH-correction via
intramuscular injection in the hind limb has been correlated with restored phenylalanine
hydroxylase levels and the proper regulation of phenylalanine levels in the blood9. The
proposed mechanism for this treatment is that the body converts ingested phenylalanine
into tyrosine, thus mimicking the normal metabolism of the amino acid in the liver of
healthy individuals7. Gene-corrected cell populations were found to aggregate in the liver,
among other organs, following this treatment. The localization of these cells in the liver
facilitates the production of metabolic enzymes necessary to break down phenylalanine.
The liver is the site of phenylalanine hydroxylase production in healthy individuals, so it
makes sense that we would see gene-corrected cell populations in this organ.
I would like to collect both quantitative and qualitative data for this experiment,
from healthy subjects as well as diseased subjects (full PAH knockout mice). There are
two treatment subgroups within the diseased population of mice. The first receives a
saline injection (this is our control) while the second group receives an injection of the

adenovirus with corrected Phenylalanine hydroxylase and tetrahydrobiopterin production

genes. 10 days after the injections, I will collect blood samples to determine PAH activity
in the blood. Tetrahydrobiopterin stabilizes PAH in the body, so adding the gene for this
component should stabilize PAH levels. I predict that my healthy population will have the
highest level of both enzymes, my treated group will have moderate enzyme levels, and
my saline group will have the lowest enzyme levels (fig. 1).
3
2.5
2
1.5
PAH Activity (mU/mL)

1
0.5
0

Figure 1: Predicted PAH Activity in

Treated vs. Control Mice 10 Days
Post-Treatment
WT Levels are elevated in the blood
of treated mice compared to nontreated mice in the PAH knockout
group. The treatment group is still shy
of normal PAH levels. Ideally, with
time, the levels in the treatment mice
will rise to normal levels.

Next, I will collect cell samples from the injection site as well as from the liver in
all groups prior to treatment, 10 days post treatment, and 1 year post treatment. I predict
that the healthy individuals will have the highest gene-corrected cell population density in
the both sites (since all of their cells contain the wild type gene). The treatment group will
have a low/moderate corrected cell density 10 days post treatment, and a relatively high
density 1 year post treatment. The saline group will have low (or zero) corrected cell
density at all checkpoints. Figure 2 shows predictions for cell populations collected from
the liver.
I will also collect data regarding phenylalanine tolerance in all animals, as
measured by symptom flare-ups in subjects. Phenylalanine will be fed to subjects at a
steadily increasing dosage until such flare ups are noted. These flare ups might include

behavioral changes, skin/hair abnormalities, or tremors/seizures. The severity of the

symptoms will be scored. The highest level of intolerance (showing the most extreme
symptoms) will receive a score of 0 while normal tolerance will receive a score of 100.
This data will be collected in tandem with the liver cell samples explained above. I am
collecting these data at the same time and from the same individuals because I am
interested in the correlation between the density of gene-corrected cells and
phenylalanine tolerance. I predict that there will be a positive correlation between these
two variables. Ideally, after a year post-treatment, the treated PAH knockout group will
show normal levels of phenylalanine tolerance, even without 100% gene-corrected cell
integration in the liver and injection site. I predict that there is a threshold of enzyme
levels below that of normal individuals that will be sufficient to produce normal levels of
phenylalanine tolerance (fig. 2).
120
100
80
60
PAH-corrected Cell Density
Column1
(%)

40

Phe. Tolerance

20
0

Figure 2: Gene corrected cell populations and Phenylalanine Tolerance in Treated vs.
Control Cells. The population of gene-corrected or WT PAH gene cells in the liver
increase in treated cells over time, though dont ever reach 100%. Phenylalanine
Tolerance is scored from 0-100, with 0 being no tolerance and 100 being normal

tolerance. This score increases in treated group. After 1 year, tolerance reaches normal
levels.
Further issues regarding this treatment plan include the disparity between its
efficacy in males and females. In mice, the treatment is much more successful and better
retained in males than in females. Furthermore, it will be some time before we know the
long-term effects of this type of treatment. This lack of data makes it risky to implement
this type of treatment on human subjects. While my proposed mechanism shows great
promise for the treatment of mild to severe Phenylketonuria, there are still discrepancies
to conduct further research on before human trials are to begin.

Works Cited

1) Christ, Shawn E., et al. "Executive function in early-treated phenylketonuria:

profile and underlying mechanisms." Molecular genetics and metabolism 99
(2010): S22-S32.
2) Acosta, Phyllis B., et al. "Nutrient intakes and physical growth of children with
phenylketonuria undergoing nutrition therapy." Journal of the American Dietetic
Association 103.9 (2003): 1167-1173.
3) Feillet, Franois, and Carlo Agostoni. "Nutritional issues in treating
phenylketonuria." Journal of inherited metabolic disease 33.6 (2010): 659-664.
4) Matalon, R., et al. "Large neutral amino acids in the treatment of phenylketonuria
(PKU)." Journal of inherited metabolic disease 29.6 (2006): 732-738.
5) Fiori, Laura, et al. "Incidence of BH 4-responsiveness in phenylalaninehydroxylase-deficient Italian patients." Molecular genetics and metabolism 86
(2005): 67-74.
6) "Phenylketonuria." Genetics Home Reference. N.p., n.d. Web. 01 Mar. 2015.
7) Strisciuglio, Pietro, and Daniela Concolino. "New strategies for the treatment of
Phenylketonuria (PKU)." Metabolites 4.4 (2014): 1007-1017.
8) Alexander, I. E., et al. "Potential of AAV vectors in the treatment of metabolic
disease." Gene therapy 15.11 (2008): 831-839.
9) Rebuffat, Alexandre, et al. "Comparison of adeno-associated virus pseudotype 1,
2, and 8 vectors administered by intramuscular injection in the treatment of
murine phenylketonuria." Human gene therapy 21.4 (2010): 463-477.