Expert Opinion on Orphan Drugs

ISSN: (Print) 2167-8707 (Online) Journal homepage: http://www.tandfonline.com/loi/ieod20

Advances in understanding disease mechanisms

and potential treatments for Crigler-Najjar
syndrome

Giulia Bortolussi & Andrés Fernando Muro

To cite this article: Giulia Bortolussi & Andrés Fernando Muro (2018): Advances in understanding
disease mechanisms and potential treatments for Crigler-Najjar syndrome, Expert Opinion on
Orphan Drugs, DOI: 10.1080/21678707.2018.1495558

To link to this article: https://doi.org/10.1080/21678707.2018.1495558

Accepted author version posted online: 03

Jul 2018.

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Publisher: Taylor & Francis

Journal: Expert Opinion on Orphan Drugs

DOI: 10.1080/21678707.2018.1495558
"Advances in understanding disease mechanism and potential treatments for
Crigler-Najjar syndrome"

Bortolussi, Giulia and Muro, Andrés Fernando

International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano, 99

– 34149 – Trieste, Italy

Corresponding author:

Andrés F. Muro
ICGEB, Padriciano 99, 34149-Trieste, TS, Italy
Telephone number: +39-040-3757369
Fax number: +39-040-226555
Email address: muro@icgeb.org

Page 1 of 45
Advances in understanding disease mechanisms and potential treatments for
Crigler-Najjar syndrome

Giulia Bortolussi and Muro, Andrés Fernando

International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano, 99

– 34149 – Trieste, Italy

Corresponding author:

Andrés F. Muro
ICGEB, Padriciano 99, 34149-Trieste, TS, Italy
Telephone number: +39-040-3757369
Fax number: +39-040-226555
Email address: muro@icgeb.org

Page 2 of 45
Abstract

Introduction: The Crigler-Najjar syndrome is an ultra-rare recessive disorder of the

liver. There is no effective cure, except for liver transplantation, a procedure with many
risks and shortcomings. Thus, the development of novel therapeutic approaches is a
clinical need.
Areas covered: This review aims to describe the potential and limitations of novel
experimental treatments that can be applied to cure the Crigler-Najjar syndrome and other
metabolic liver diseases. These include pharmacological and gene therapy approaches,
some of them are now being applied in clinical trials of Crigler-Najjar patients. Brief
descriptions of bilirubin metabolism, mechanisms of disease and animal models are also
included.
Expert Opinion: AAV-mediated gene therapy approaches to pediatric liver diseases are
very challenging due to the potential loss of viral DNA consequent to liver growth. Novel
immunomodulatory strategies are being developed that can allow re-administration of the
therapeutic vector by blocking the generation of anti-AAV neutralizing antibodies.
Another very promising approach is gene targeting of the therapeutic cDNA into a “safe-
harbor locus”, resulting in the permanent correction of the genetic defect. However,
further experimentation is still required to increase safety and efficacy. The success of
these approaches will result in alternative therapies to liver transplantation.

Keywords: kernicterus, phototherapy, bilirubin-induced neuro-inflammation, AAV-

mediated liver gene therapy, genome editing

Financial and competing disclosure: The authors report no conflicts of interest

Page 3 of 45
1. Bilirubin metabolism at a glance.
Bilirubin is the catabolic product of the protoporphyrin portion of haeme groups present
mainly in hemoglobin, but also in myoglobin and haeme-containing enzymes, such as
cytochrome P-450 1. This process, necessary to eliminate waste products generated from
the destruction of cells and cellular components, involves two enzymatic reactions.
Briefly, the heme group is cleaved by microsomal heme-oxigenase 1 (HO-1) to generate
biliverdin (Figure 1A). Next, biliverdin is further reduced to unconjugated bilirubin
(UCB) by biliverdin reductase (BVR).
UCB, a highly hydrophobic molecule with high affinity for lipid-rich tissues and
albumin, is transported via the circulation to the liver bound to albumin, where it passes
across the sinusoidal-hepatocyte interface by a bi-directional flux. UCB enters the
hepatocyte by facilitated diffusion where it is bound to cytosolic proteins such as
ligandin, and reaches the endoplasmic reticulum where it undergoes a double-step
reaction in which two glucuronic acids are conjugated to its two propionyl side chains.
By this enzymatic modification bilirubin solubility is increased and can easily pass into
the bile by means of the active transporters Mrp2 (ABCC2) and, at a minor extent, Bcrp1
2, 3
(ABCG2) (Figure 1B). The bile is further released into the gastro-intestinal tract,
where intestinal bacteria reduce conjugated bilirubin to colorless pigments named
urobilinogens. Next, these pigments are converted to urobilins and eliminated with feces
and urine as sterco- and urobilins, respectively. A variable fraction (4-25%) is re-
adsorbed by the intestine after being de-conjugated by bacterial enzymes and transported
back to the liver in a process known as enteropatic circulation 4-6 .

2. The UGT1A1 enzyme.

UDP-glucuronosyl transferase proteins (UGTs) belong to a superfamily of enzymes that
catalyze the clearance of environmental toxins, endogenous metabolites and therapeutic
drugs by means of glucuronidation. Based on their homology and evolutionary
divergence, the UGT family has been divided in two main families: UGT1 and UGT2 7.
While independent genes codify for the UGT2 family, UGT1 diversity originates from a
peculiar genomic organization. In fact, the UGT1A gene cluster, located in chromosome
2-q37, has 13 different first exclusive exons and promoters (UGT1A1 to 13p) encoding

Page 4 of 45
the amino-terminal substrate binding domain, and four common exons encoding the
8, 9
active domain and the transmembrane domain . Thus, UGT1 enzymes share an
identical carboxy-terminal codifying for the endoplasmic reticulum-binding membrane
domain and the binding for the co-substrate, while their N-terminal portion is substrate-
specific.
Among these enzymes, the uridine diphospho-glucuronosyltransferase 1A1 (UGT1A1)
10
enzyme is the only one responsible for the bilirubin-glucuronidation activity , and its
defect results in increased systemic levels of UCB. It is mainly expressed in liver
although the intestine, kidney and skin show minor levels of expression 11, 12.

3. The unconjugated hyperbilirubinemias.

Mutations in the common or in the bilirubin-specific regions of the UGT1A1 gene,
affecting its expression and/or activity, lead to different forms of unconjugated
bilirubinemia. The diversity in the nature of these mutations results in a range of
phenotypes, extending from mild (Gilbert syndrome) to very severe (Crigler-Najjar
syndrome type I) 13-15, as described below (Table 1 and Figure 2).
An updated list of disease-causing mutations of Gilbert and Crigler-Najjar syndrome,
accounting for more than 130 different cases, was recently published 13. The majority of
these mutations (about 70%) are single nucleotide substitutions, while deletions,
insertions, and mutations affecting the promoter region account for the remaining 30%
(see Canu et al 13 for details).

3.1. Gilbert syndrome (GS) is the most benign form of hereditary

unconjugated hyperbilirubinemia and these patients carry a normal life. They present
total serum bilirubin levels rarely exceeding 4 mg/dl, while the liver and bile acid test are
normal. Although total serum bilirubin levels may rise during starvation or upon hepatic
16
complications, GS patients have almost no risk of developing brain damage .
Additionally, several studies have shown that GS patients have less probability of
developing atherosclerosis, cardiovascular disease, coronary heart disease and myocardial
infarction 17, 18, due to the anti-inflammatory and antioxidant properties of UCB at mildly
elevated concentrations 19.

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The most common genetic mutation of GS is a polymorphism in the TATA box region of
the UGT1A1 promoter region [A(TA)7TAA, instead of A(TA)6TAA] (also known as
UGT1A1*28), causing a significant reduction of UGT1A1 transcriptional activity 20. This
polymorphism is very frequent in the general population, with prevalence ranging from
21
10% to 20%, depending on the ethnic group . To note, the presence of the
polymorphism in the genome is not sufficient for complete manifestation of the disease,
indicating that more factors affect the clinical outcome of the GS patient. Other mutations
are point missense mutations resulting in an important reduction of enzyme levels or
activity 13.

3.2. The Crigler-Najjar syndrome (CNS). Crigler and Najjar were the first to
characterize the syndrome in 1952, as severe congenital familiar non-hemolytic jaundice
22
. The Crigler-Najjar syndrome (CNS) is a recessively inherited metabolic disorder of
the liver with an estimated frequency of 1 in a million live births and is equally
distributed in both genders 23, 24. It has been reported that CNS is relatively more frequent
in specific populations such as the Amish and Mennonite communities, and in the sub-
25, 26
Mediterranean region (Tunis) . Two distinct forms of the disease have been
characterized according to the severity of the outcome: type I (CNS-I) and type II (CNS-
II). Untreated CNS-I patients have total serum bilirubin levels exceeding 20 mg/dl
(340µM) and can increase up to 50 mg/dl (850µM). This is the most severe form of the
disease since patients completely lack UGT1A1 enzyme activity. Bile acid test in these
patients showed that bile is almost completely composed by unconjugated bilirubin with
27
only traces of mono-glucuronide bilirubin . If not promptly treated, these patients
develop severe neurological damage and are at constant risk of kernicterus. Genetic
mutations in CNS-I patients are well distributed along all the exons and are point
missense or nonsense mutations, single base deletions, insertions or in few cases
promoter polymorphisms (see 13 for a detailed description).
In contrast, CNS-II patients have total serum bilirubin levels in the range of 3.5-20 mg/dl
(60-340 µM). This syndrome is also known as Arias syndrome and shows a better
outcome 28. In fact, these patients have a residual UGT1A1 enzymatic activity (less that
10%), which is sufficient to maintain UCB levels below the risk of developing serious

Page 6 of 45
neurological damage. UGT1A1 enzymatic levels can be increased by treatment with
phenobarbital, which stimulates UGT1A1 gene transcription. This pharmacological
treatment is usually used to discriminate between type I and type II CNS forms 28. Type
II syndrome is caused by homozygous or compound heterozygous mutations in the
UGT1A1 gene. It has been reported that some patients carry a combination of
heterozygous CNS and Gilbert mutations (see 13 for a detailed list).

4. The Neurological outcome.

UCB travels in the bloodstream bound to albumin and, in normal conditions, the amount
29
of unbound UCB to albumin is negligible (less than 0.1%) . When UCB levels exceed
the albumin binding capacity, the fraction of unbound UCB, also known as free bilirubin
(Bf), increases and accumulates in lipophilic tissues such as the brain, causing
neurological damage 30.
Milder and reversible forms of neurological damage include bilirubin-induced
neurological dysfunction (BIND), with hypotonia, lethargy, anorexia and abnormal
auditory brainstem evoked potentials (ABRs). In more severe cases patients develop
moderate movement disorders such as abnormal gait, imbalance, impaired fine motor
skills and ataxia due to the deposition of bilirubin in the basal ganglia and cerebellum.
The most severe, irreversible form is kernicterus. The term refers to the anatomic
diagnosis made at autopsy, based on the pattern of brain staining and literally indicates
“yellow nuclei”. In fact, bilirubin deposition in the brain occurs mainly in basal ganglia,
globus pallidus, hippocampus, subtalamic nucleus, horn of Ammon, cranial nerve nuclei
31
and the cerebellum . Choroathetoid cerebral palsy, high-frequency central neural
hearing loss, palsy or vertical gaze and dental enamel hypoplasia are main features of
kernicterus. The prognosis of kernicterus is unfortunately very poor and may lead to
death, if untreated.

5. Animal models of congenital unconjugated hyperbilirubinemia

Different animal models have been used to study the in vivo mechanisms of disease and
to develop new therapeutic approaches. The Gunn rat is by far the most widely used
hyperbilirubinemic model 32, 33. This animal strain has a spontaneous one-base deletion in

Page 7 of 45
the exon 4 of the Ugt1 locus, creating an in-frame premature stop codon, that is translated
into a truncated protein lacking the transmembrane domain (which is rapidly degraded)
33, 34
and deficiency of all members of the Ugt1a isoenzymes . Those animals reproduce
important features of the human CNS-I such as life-long severe non-hemolytic
35
unconjugated hyperbilirubinemia, but their phenotype is mild . In fact, untreated
homozygous Gunn rats present cerebellar abnormalities and hearing impairment, but
35
reach adulthood and are fertile , without fully reproducing the human CNS-I. They
develop acute central nervous system dysfunction and eventually kernicterus only when
treated with UCB-albumin displacers (such as sulphonamides) or with erythrocyte-lysing
agents such as phenylhydrazine 36. Despite these limitations, they have been widely used
and provided important clues in the bilirubin field ranging from toxicological studies, to
neuroscience and gene therapy.
Still, the higher availability of mutant mouse strains and the possibility to manipulate the
mouse genome poses limits to the use of the Gunn rats to study the participation of other
genes in the regulation of UCB metabolism and its toxicity, or the effects of moderate
UCB levels in the development of pathologies such as atherosclerosis, cardiovascular
disease and cancer.
Mouse models containing null mutations and conditional mutations of the Ugt1 gene
were generated by two different laboratories: a) a drastic disruption of the Ugt1 gene by
insertion of the neomycin cassette 37, b) a one-base deletion generating a frame-shift and
38
a premature stop codon, identical to the one present in Gunn rats , c) tissue-specific
39
Ugt1 locus conditional knockout mouse models , and d) a humanized mouse model
containing the UGT1A1*28 allele in Ugt1 null genetic background reproducing main
features of the Gilbert’s syndrome 40.
Unexpectedly, both mouse models bearing null mutations displayed a much stronger
phenotype than the one observed in the Gunn rats, as all mutant mice died within a few
37, 38
days after birth due to UCB toxicity . Indeed, these mouse models more closely
reproduce the human features of CNS-I such as motor coordination impairment,
37, 38
neurological damage and ultimately death . The reasons for these differences are not
yet clear, as bilirubin levels in mutant rats and mice appear to be within a similar range 41-
44
, but parallels the results obtained when the Ugt1a mutation was studied in different

Page 8 of 45
genetic backgrounds of mice, as it emerges that susceptibility to bilirubin damage and,
44
consequently, survival of mutant mice is strain-specific , supporting the hypothesis of
modifier genes modulating bilirubin neurotoxicity 30.

6. Mechanisms of neurological damage.

The genetic and molecular mechanisms leading to bilirubin accumulation and cellular
events related to its toxicity have been extensively studied during the past decades with
the aim of an early diagnosis and better treatment for the disease. Several in vitro studies
reported that excessive UCB levels induce oxidative stress, affect mitochondria
45-47
metabolism and glutathione homeostasis . In addition, immature neurons show
increased susceptibility to bilirubin toxicity compared to more differentiated ones, having
48, 49
impaired neurogenesis, and cell death . Recent studies in immortalized cell lines
50
showed that elevated levels of UCB induce ER stress response and autophagy . Cell
culture studies using primary microglia cells dosed with bilirubin indicate that
inflammation is increased upon bilirubin exposure and that old microglia is less efficient
than the younger one in responding to the bilirubin insult 51-53.

6.1. Neurotoxicity and neuroinflammation in vivo. The elucidation of the

molecular events associated with neurological damage experienced a new flow of
investigation after the gene targeting development. In fact, before it the possibility to
study the functional effect of genes relied only in spontaneous mutations isolated from
living stocks (such as the Gunn rat) and the modulation of UCB levels using bilirubin
displacers (sulphadimethoxine) or drugs inducing hemolysis (phenyhydrazine), as
mentioned above. Thus, the easy to manipulate the mouse genome allowed the generation
of several mouse models of unconjugated hyperbilirubinemia and bilirubin detoxification
37, 38, 40, 44, 54
. Thanks to these new mouse models, the understanding and the management
of the disease has improved significantly (see also Section 8.1, Potential Pharmacological
Treatments below for further details).
In particular, these models shed light on the mechanism underling neurological damage.
It was shown that susceptibility to bilirubin damage is strain specific in mice, supporting
the study of genes modulating the severity of the disease (see Section 6.2 below).

Page 9 of 45
In addition, bilirubin toxicity is time-dependent and affects predominantly poorly
44 55
differentiated or immature neurons , with an impact in myelin sheath formation .
Consistent with previous findings in Gunn rats, Purkinje cells and Granule cell precursors
in the cerebellum of mutant mice were the most susceptible cells of the developing brain
44, 56, 57
. Moreover, a proteomics analysis showed that bilirubin injury affects antioxidant
defenses in the cerebellum of the Ugt1-/- mice, reducing the overall capacity of neurons to
overcame neurological damage 56.
Finally, the importance of ER stress and inflammatory response upon bilirubin exposure
57, 58
has been reported . In particular, it was observed that in hUgt1/TLR2-/- mice the
absence of inflammatory response reduced survival of the double KO animals, indicating
that a certain level of inflammation improves the outcome. On the other hand, treatment
with a neuroprotective and anti-inflammatory agent (minocycline, MNC) increased
survival and reduced neurological damage in Ugt1-/- neonatal mice, without affecting
59
total bilirubin levels . MNC-rescued mice showed normal motor-coordination
performance and behavior, reduced ER stress and activation of neurotoxic microglia,
resulting in an improvement in dendritic arborization of Purkinje cells. These results
underscore the importance of the modulation of the inflammatory response to overcome
bilirubin neurotoxicity and support further pharmacological studies to modulate these
pathways.

6.2. Bilirubin transport in the brain. In addition to differences in phenotype

severity among strains of rodents having the same genetic mutation, huge variability in
the extent of neurological damage was also observed at similar levels of bilirubin in
33, 38, 44
human newborns . The variability in the extent of bilirubin-induced neurological
damage is still poorly understood and further studies will help to discover the molecular
mechanisms involved in its modulation, resulting in the identification of novel genes and
unexpected potential therapies. Candidate genes belonging to the ABC super-family of
60-62
transporters have been identified using in vitro cultures of neuronal cells . These
transporters [ABCB1/P-glycoprotein (P-gp) and ABCC (multidrug resistance-associated
proteins, MRPs; ABCC1/ MRP1)] have been suggested to actively export unconjugated
bilirubin from the central nervous system into plasma and cerebro-spinal fluid (CSF),

Page 10 of 45
 30, 46, 63
consequently modulating the risk of developing bilirubin-induced brain damage .
However, when these bilirubin transporters were evaluated for their ability to modulate
bilirubin toxicity in the brain using engineered mouse models, only hyperbilirubinemic
mice lacking Abcb1 died, indicating that this transporter is essential to protect the
cerebellum from bilirubin toxicity during neonatal development 54, the most critical phase
for human babies.

7. Current treatments to evade hyperbilirubinemia and neurological damage.

Before the discovery of phototherapy, survival of severe Crigler-Najjar patients was very
22, 64
limited, with death frequently during infancy between the age of 1 and 2 years . The
management of the disease was considerably improved upon discovery that exposure to
65, 66
sunlight reduced bilirubin levels in jaundiced babies , due to the isomerization of
UCB into water-soluble photo-isomers that are excreted in bile and urine 67.
Presently, affected patients are treated with whole-body exposure to intensive
phototherapy (PT) for up to 10 to 12 hours/day. In acute situations PT is combined with
exchange transfusions to rapidly reduce peaks of plasma bilirubin.
PT, although effective in the first years of life, affects significantly life quality and has
important limitations, such as gradual loss of efficacy over time caused by the decrease in
the surface/volume ratio and the thickening of the skin during growth, also consequent to
the intensive PT. Thus, patients need more intense and prolonged PT, without eliminating
the risk of life threatening spikes of UCB (for instance, during trauma or sepsis) and the
development of bilirubin encephalopathy and permanent brain damage later in life 68.
Daily Crigler-Najjar syndrome treatment medications may include phenobarbital and
ursodiol 69, 70, while vitamin E, vitamin C, coenzyme Q, L-carnitine, and creatine may be
71-76
supplemented for their neuroprotective properties . When unconjugated bilirubin
reaches toxic levels, the disease is managed with aggressive intravenous fluid hydration,
administration of albumin, and possibly plasma exchange to avoid serious neurological
consequences. Albumin infusion increases the bilirubin plasma-binding capacity
capturing bilirubin excess and, thus, lowering the total body exchangeable UCB fraction
and preventing its movement and accumulation in extravascular sites. Its use is accepted
25, 77-80
in many clinical centers . Ursodiol, lipid-rich food, agar or oral calcium carbonate

Page 11 of 45
may be given to increase the intestinal flow or to trap bilirubin in the intestinal lumen,
25
maximizing elimination of UCB and derivatives with the feces . However, those
treatments present significant limitations and may have, in some cases, important risks
and undesired side effects. Therefore, most patients suffering from the severe form of
CNS-I do need liver transplantation at some point in their life. Orthotopic liver
transplantation (OLT) to restore UGT1A1 activity is still the only definitive cure for the
24, 25
severe forms of CN syndrome . Regrettably, OLT is a very invasive procedure with
substantial risks (5-10% patient mortality within the first 5 years after liver transplant 81)
and important shortcomings such as: organ incompatibility/rejection and need of re-
transplantation, which is not possible in all cases due to shortage of compatible organs;
life-long immunosuppression to prevent liver rejection; increased risk of skin and
lymphoproliferative tumours 82-84.
For these reasons, the current therapeutic approach is suboptimal and the development of
safer and more effective treatments for CNS is a clinical need. Nowadays, novel curative
options based on gene therapy appear to be valid therapeutic approaches for definitively
curing the CN syndrome (Table 2).

8. Potential treatments for Crigler-Najjar syndrome

We can roughly divide the potential treatments in two main categories (Table 2 and
Figure 3):
- Those aiming to control bilirubin and its neurotoxic effects: pharmacological
treatments
- Those aiming to restore UGT1A1 activity in hepatocytes: cell and gene therapies

8.1. Potential pharmacological treatments. To the first category belong

proof-of-concept studies blocking the production of biliverdin from heme groups with an
inhibitor of heme oxygenase-1 (HO-1), obtained by treating neonatal hyperbilirubinemic
85
mice with zinc protoporphyrin . Treated animals had a reduction in plasma levels of
bilirubin and were rescued from bilirubin-induced neonatal lethality. An alternative
approach could be the blockade of biliverdin reductase with specific and potent

Page 12 of 45
inhibitors, although potential candidates selected from an in vitro screening did not show
the expected result so far 86.
87, 88
Mutations in the HO-1 gene result in a lethal disease in children and caused serious
89
problems in knockout mice , raising an important concern about the chronic treatment
with HO-1 inhibitors, suggesting that further studies are required. On the contrary,
patients and mice bearing mutations in the biliverdin reductase alpha gene (BLVRA) live
a normal life 90, 91. The potential use of biliverdin reductase inhibitors is also supported by
the observation that high biliverdin levels are not toxic, as demonstrated by several
animal species that do not have BLVRA activity and adopted biliverdin as the main
heme-degradation product 92.
Another approach aiming to provide a fast and effective temporal treatment reducing the
risks of bilirubin-induced brain damage in acute events resides in the administration of
albumin to capture the excess of free bilirubin in plasma, forcing the mobilization of
36, 93, 94
accumulated bilirubin from tissues to plasma . This approach is occasionally used
prior to exchange transfusions (usual dose is 1-2 g/kg/dose, administered i.v. every 12 h
25 80, 95
, although higher doses have not resulted in adverse effects ) and has shown
79
benefits to reduce auditory damage in hyperbilirubinemic children . Recently, albumin
administration experiments were performed in Gunn rats and Ugt1-/- mice. When a single
dose of albumin was administered to Gunn rats, the plasma Bf was significantly reduced
93
and it protected them against bilirubin-induced neurological damage 36. Daily albumin
administration since birth to Ugt1-/- pups completely rescued neurological damage and
94
lethality . These results support the potential use of albumin administration in severe
acute hyperbilirubinemia conditions, to prevent or limit bilirubin neurotoxicity.
Regrettably, in spite of the benefits observed in animal models and patients, its use as a
treatment for acute hyperbilirubinemia is not yet widely adopted in the clinic.
During hyperbilirubinemia, an important proportion of plasma UCB reaches the intestinal
lumen via diffusion across the intestinal mucosa, but a fraction is reabsorbed. Therefore,
experimental therapies focused in preventing reabsorption of bilirubin from the intestinal
lumen have been tested and were effective to produce a minor decrease in plasma
bilirubin levels in Gunn rats and patients (about 10%). However, these treatments have

Page 13 of 45
not been adopted as they present important side effects and high therapeutic variability 69,
96, 97
.
Proof-of-concept studies showing the reduction in neurodegeneration and partial rescue
of neonatal lethality of Ugt1-/- mice by the treatment with minocycline, a second-
generation tetracycline with potent anti-inflammatory properties were recently published
59
. However, the use of this compound is limited by its photo-sensibility and by several
98, 99
side effects , prompting the search for effective anti-inflammatory drugs without
undesired side effects. Derivatives of MNC have been proposed, such as PMIN and
COL3, but results were not conclusive 100, 101. Nevertheless, these results pave the way to
find other compounds with strong neuroprotective and anti-inflammatory properties,
devoid of undesired side effects that could be administered in combination with
phototherapy.

8.2. Hepatocyte and stem cell transplantation. Therapies based on the

transplantation of allogeneic hepatocytes or hepatocyte progenitor cells have the potential
to cure inherited liver disorders. They are attractive approaches since they are minimally
invasive, can be performed repeatedly if required, the native liver is not removed, and can
greatly reduce the risk of fatal complications of whole organ transplantation 81. However,
a number of limitations still exist, such as the low levels of cell engraftment, expansion
and maintenance of cellular viability post-transplant, potential immune responses leading
to rejection or graft vs. host disease, and increased risk of tumor associated to prolonged
immunosuppression 82-84.
Long-term rescue of hyperbilirubinemia was obtained by the infusion of human and rat
102, 103
hepatocytes into Gunn rats , (see Lysy et al for additional references in liver cell
transplantation 104), providing experimental support to the application of this approach in
the clinics. More recently, the use of adult stem/progenitor cells and iPS cells has
emerged as alternative cell sources for hepatocyte transplantation due to the possibility of
“unlimited” supply of hepatocytes 105.
Transplantation of isolated allogenic hepatocytes has been attempted in Crigler-Najjar
106-108
patients, however achieving only limited and transient benefit . The poor levels of
engraftment of transplanted cells and the lack of growth advantage of the transplanted

Page 14 of 45
cells resulted in a decline in cell function after 9-11 months, requiring liver
106
transplantation . Similar results were also shown in a recent cell transplantation trial
109
with mesenchymal stem cells . Different approaches have been used in animal models
to increase engraftment rate, ranging from partial hepatectomy, irradiation, CCl4
110-112
treatment, to block of endogenous hepatocyte proliferation . However, these
treatments cannot be applied to patients and safer strategies need to be developed.
Consequently, hepatocyte transplantation in Crigler-Najjar patients is performed as a
temporary therapeutic alternative approach or “bridge” for patients waiting for whole
organ transplantation.

8.3. Gene therapy for metabolic liver diseases. Very promising alternative
approaches that may provide long-term correction of the genetic defects are gene therapy
and gene editing.
Gene therapy by gene replacement consists in the addition of new genes to a patient's
cells to replace missing or malfunctioning ones.
Recombinant adeno-associated virus (AAV) vectors have emerged as the most promising
gene delivery systems for gene replacement therapy of monogenic liver disorders, having
an excellent benefit-risk ratio 113. They have been successfully employed in clinical trials
114-117
of hemophilia A and B (coagulation factor VIII and IX deficiencies, respectively) ,
and are the subject of numerous ongoing clinical trials (http://www.clinicaltrials.gov). In
this approach, the therapeutic cDNA remains as an episome in the diseased cell and its
transcription is controlled by a liver-specific promoter. However, their application during
infancy or juvenile age to treat diseases in which the target organ is growing is still
limited by concerns related to safety and long-term efficacy of the therapy, concerns that
require further experimentation.
In fact, when rAAV is delivered during the neonatal or juvenile age to the liver,
consequent to the episomal nature of the viral DNA, there is a progressive loss of viral
genomes and transgene expression over time in concert with hepatocyte proliferation and,
118-120
consequently, therapeutic efficacy . In the case of AAV-mediated gene therapy of
genetic liver diseases, this feature is of particular importance for the treatment of
paediatric subjects, or in the context of specific disease states causing hepatocyte

Page 15 of 45
proliferation in adult AAV-treated patients. Re-administration of the therapeutic vector is
prevented by the presence of anti-AAV capsid proteins, generated after the first
administration. The vector dose-dependent (re)activation of these immune responses can
120, 121
result in clearance of transduced cells and loss of transgene expression . A possible
approach to overcome this problem is the co-treatment with immune-modulators that may
block or limit the generation of neutralizing antibodies (Nabs) at the moment of the first
AAV administration. A very promising candidate is rapamycin encapsulated in synthetic
122,
nanoparticles, which was shown to induce antigen-specific immunological tolerance
123
.
The efficacy of AAV-mediated gene therapy could be reduced by the presence of pre-
existing anti-capsid neutralizing antibodies in those patients exposed to the virus from
which the vector is derived, which can prevent the efficient transduction of the target
organ 124, 125. Neutralizing antibodies against the different AAV serotypes are found in the
population with prevalence reaching 60% for AAV2 and 20% for AAV8 126 thus, limiting
the enrolment of patients in gene therapy trials.
Immunity against the therapeutic protein is a general concern of the gene therapy field, as
this may hinder the long-term efficacy of the treatment. However, the liver can be
considered a privileged organ as studies using AAV-mediated gene therapy in small and
large animals, and in humans, resulted in multi-year expression of the transgene with the
induction of antigen-specific tolerance 127-130.
Integration of recombinant AAV vectors into the host genome occurs at a low frequency
into random sites 131, 132. Reports addressing genotoxicity following AAV gene transfer in
mice are contrasting 133-135. On the contrary, multi-year data in large animal models 136, 137
116, 117, 137
and humans suggest that the risk of genotoxicity following rAAV
administration is minimal. However, a recent report showing the association of wild-type
AAV2 with oncogenic insertional mutagenesis in human HCC suggest that a long-term
follow up of patients will be required to address these potential concerns 138.
Systemic delivery of the therapeutic mRNA as biodegradable lipid nanoparticles (LNPs)
is a promising strategy that overcomes pre-existing immunity and potential genotoxicity
of AAV vectors. However, the long-term application of this strategy may be limited by
the transient expression of the transgene, requiring very frequent re-administration of the

Page 16 of 45
 139-141
LNPs . LNPs application could certainly represent an important alternative therapy
for liver diseases in the case of acute metabolic decompensations, which require
immediate treatment to avoid death and prevent associated complications.

8.4. Viral gene therapy for CN syndrome. The availability of relevant

animal models of the disease is an essential requirement for the development of effective
therapies. The Gunn rats were used to test numerous gene therapy approaches, ranging
from naked plasmid administration, to chimeric oligonucleotides, adenoviral-, lentiviral-
42, 43, 142-148
and AAV-vectors . These experiments provided the proof-of-principle that
gene therapy for Crigler-Najjar resulted in liver glucuronidation activity, the presence of
conjugated bilirubin in bile and an important reduction of plasma bilirubin, but the real
proof of efficacy in a severe animal model with neonatal lethality, as observed in
untreated CNS-I patients, was still missing.
To this purpose, AAV-mediated gene therapy approaches were tested in neonatal mice
bearing null mutations in the Ugt1 locus, animals that do not survive beyond the first
37, 38, 44
week of life . Treatment of those animals with AAV vectors encoding WT and
codon optimised versions of the hUGT1A1 cDNA with liver-specific expression of the
118, 149
transgene resulted in the full rescue the lethal phenotype . However, long-term
protection of the mutant animals after neonatal delivery was only obtained with very high
118, 149, 150
doses of the vector, in the range of 5.0E13 vg/kg or higher , raising important
safety concerns. Importantly, the optimization of the UGT1A1 expression cassette
allowed the administration of much lower AAV doses in adult Gunn rats and neonatal
Ugt1-/- mice (5.0E11 vg/kg and 8.0E9 vg/mouse, respectively), resulting in the long-term
correction of the phenotype only in rats 145 These data supported the use of this vector in
a clinical trial for Crigler-Najjar. Today, two AAV8-mediated clinical trials are being
performed in Crigler-Najjar patients, but no results are available so far. One relevant
consideration is that in the case the AAV-mediated gene therapy results in insufficient
correction of the disease phenotype or there is an important loss of efficacy over time,
treated patients will still be able to access phototherapy and will still be eligible for liver
transplantation.

Page 17 of 45
Different approaches may be used to prevent the loss of therapeutic efficacy of gene
therapy consequent to cell proliferation. Re-administration of the AAV vector with
serotype switching to avoid the potential presence of neutralizing antibodies against the
second AAV capsid was effective in Ugt1-/- animals, suggesting the feasibility of the
approach, at least in mice 150. Re-administration of the same original vector was effective
in mice but only at a high vector dose 149. However, these successful approaches in mice
do not completely rule out the potential presence in patients of Nabs or cross-reactive
Abs generated after the first administration, suggesting that more efficient approaches
should be developed. A promising possibility regards the modulation of the immune
response at the moment of the first AAV administration using immunomodulators, such
as SVP-rapamycin nanoparticles that block the immune response and, thus, the
generation of neutralizing antibodies against the viral capsid proteins 122, 123. Nonetheless,
repeated administration of the AAV vector at high doses raises the potential risk of
150
genotoxicity . In the study of Bockor and colleagues, the presence of pre-neoplastic
nodules was observed in 15 month-old mutant animals double-injected with doses
reaching 1.0E14 vg/kg of a non-optimized AAV vector. Importantly, efforts to improve
145
vector efficacy and safety resulted in a very potent and safe vector , as mentioned
above in this section. Effectiveness of this improved vector is about 100 times higher
than the studies reported so far in adults mice, without any sign of genotoxicty, as
determined 9 months after vector administration. Thus, repeated administration of low
doses of AAV in combination with SVP-rapamycin nanoparticles will provide safety and
effectiveness in long-term therapeutic protocols.

8.5. Targeted genome integration as a potential therapy for CN syndrome.

Genome editing, defined as the specific and permanent modification of the genome in a
living organism, is a promising alternative to correct disease-causing mutations. This
approach resides in the insertion, deletion or replacement of DNA at a precise site in the
genome, which is then stably transmitted to daughter cells upon duplication. The
observation that the presence of a double strand break (DSB) in the genome enhances
151
homology-directed repair , combined with the development of engineered platforms
152
able to produce site-specific DSB in the genome , made gene therapy by genome

Page 18 of 45
 153, 154
engineering one of the fastest growing fields . Site-specific correction of disease-
causing mutations is now at the reach of our technical capacities and has been also tested
155
in animal models of disease , although the presence of numerous mutations causing
CNS poses important limitations to the clinical translation of this strategy. In fact, the
correction of each mutation requires the development of specific editing protocols and,
consequently, medical products. By this reason, many efforts are being dedicated to
safely and efficiently target a single therapeutic cDNA into a “safe harbor locus”, able to
correct most disease-causing mutations with a single experimental approach. However,
several concerns need to be addressed before the final approach of any of those strategies
to the clinics. One of the main concerns of the editing/targeting strategy is consequent to
the use of engineered nucleases (ZFN, TALEN or CRISPR/Cas9), with potential off-
target effects resulting in mutagenesis (increased by the long-term expression of the
nuclease), insertional mutagenesis by the DNA carrying the nuclease, transactivation of
nearby genes, and risk of tumorigenesis 156-158.

To overcome these issues, the group of Prof. Mark Kay and collaborators developed a
strategy based on the spontaneous recombination of a targeting vector containing a
promoterless cDNA of the therapeutic gene (in the first case it was used the cDNA of the
159
coagulation factor IX) into the albumin locus . This results in a fused mRNA
transcribed by the potent albumin promoter, that is then translated into two separate
proteins (albumin and the therapeutic cDNA), avoiding the reduction of albumin
production consequent to the transgene insertion. The low levels of spontaneous
159
recombination, about 0.5% , are compensated by the high expression of the albumin
locus, resulting in therapeutic levels of the transgene.
When the same approach was applied to a lethal mouse model of Crigler-Najjar
syndrome type I, the treatment resulted in the complete rescue of neonatal lethality,
converting a severe lethal disease into a mild form, with stable low plasma bilirubin
levels even 12 months after AAV delivery and no abnormalities in liver and brain 160.
Although this approach was very effective in mouse models, the translation of this
technology to the clinic may be limited by the low absolute targeting rate and the high
viral dose required to achieve therapeutic efficacy. Improved strategies should be also
considered for therapeutic applications, such as the enhancement of the recombination

Page 19 of 45
rate by the use of high fidelity endonucleases with temporal expression of by means of
mRNA/protein delivery, self-targeting versions and/or drug-controlled activity, in order
to minimize long-term undesired effects 161-165.

9. Expert opinion
The first obstacle to overcome in the gene therapy field is the efficient delivery of the
transgene expression cassette to the target organ, by means of a vector containing the
genetic material. The ideal vector should result in the efficient transduction of the target
tissue/cell, leading to a sufficient, long-lasting and safe expression of the therapeutic
transgene, without generating immune responses against the vector itself or the transgene.
The use of nanoparticles to deliver the genetic material present numerous advantages in
terms of ease of production yields, costs of the vector, and low immunogenicity
compared to some viral approaches. However, these methods still require further
optimization to be efficiently translated to the clinics 166.
Recombinant AAV vectors represents today the most efficient approach for introducing
genetic material into diseased liver cells. The potential of gene therapy to cure liver
diseases has been recently demonstrated in AAV-mediated clinical trials for hemophilia
A and B, liver diseases characterized by the absence of liver parenchyma damage 114-117.
Similarly, the Crigler-Najjar syndrome present many features that make this disease an
excellent candidate for clinical trials: a) it is a monogenic disease; b) it is a potential
lethal disorder; c) there is no effective treatment, except for liver transplantation, which
has numerous risks and shortcomings; d) it is a well characterized disorder; e) the
mutation results in the presence of a toxic metabolite that does not affect hepatocytes,
exerting its damaging effect in the brain (non-cell autonomous feature); f) the liver is
generally normal, with no parenchymal damage; g) partial restoration of enzyme activity
is sufficient to minimize the risk of brain damage; h) efficacy of the treatment is easy to
monitor by routine biochemical tests, although more invasive approaches such as liver
biopsy or bile collection might be indicated to obtain a direct measurement of enzyme
activity ; i) limited efficacy of the gene therapy approach, or loss of efficacy over time,
does not preclude the option of OLT. However, in spite that histopathologic
abnormalities are rare in cases of CNS-I, recent reports showed the presence of fibrosis in

Page 20 of 45
patients, increasing with age 167, 168. These results suggest that earlier intervention may be
advantageous since the presence of fibrosis may affect hepatocyte transduction and long-
term efficacy.
Two parallel initiatives are being carried out to perform clinical trials in Crigler-Najjar
patients with AAV vectors: the CureCN consortium, coordinated by Dr. F. Mingozzi
(clinicaltrials.gov: NCT03466463), and one developed by Audentes Therapeutics, USA
(clinicaltrials.gov: NCT03223194). Both initiatives are based on the intravenous
administration of a rAAV8 vector carrying codon-optimized versions of the human
UGT1A1 cDNA, under the control of liver specific promoters, while cohort distribution
of patients and ages differ (Table 3).
Other key challenges of the gene therapy field are: a) re-administration of the therapeutic
vector in the case of loss of activity due to dilution of the episomal viral DNA during
hepatocyte proliferation, and b) administration of the therapeutic vectors to patients with
natural immunity against the capsid of the therapeutic vector. In the first case, treatment
of young pediatric patients is limited by the expected loss of activity, and a successive re-
administration is prevented by the production of neutralizing antibodies, generated after
the first administration. Immunomodulatory strategies may be used prevent the
development of neutralizing antibodies. This strategy may extend the application of the
present AAV-mediated therapy to young pediatric patients, also of other liver disease in
which no effective alternatives are available, such as severe urea cycle diseases. In the
second case, the selective removal of anti-AAV neutralizing antibodies from the patients’
bloodstream may allow their enrolment in current or future clinical trials. Both challenges
are being addressed by the CureCN consortium, which is composed by European
research groups, patient’s association, and biotechnology companies. It is expected that
these technologies will be available in the near future, in order to make the treatment
available to all patients.
Integrative gene therapy is a very promising possibility to cure liver genetic diseases.
This methodology will allow the permanent correction of the genetic defect in pediatric
patients, since the correction is stably transmitted to daughter cells during hepatocyte
proliferation, with no loss of therapeutic efficacy. However, these approaches are
preferentially focused in the insertion of the curative cDNA into a “safe harbor locus”, in

Page 21 of 45
order to develop a unique curative strategy covering most genetic defects. This
methodology is being developed for hemophilia B using zinc finger nucleases to increase
the rate of gene targeting, supported by pre-clinical data showing the potential of the
approach 169, 170.
Similar approaches for the Crigler Najjar syndrome are being developed using the gene-
159
ride technology , without the use of nucleases to integrate the transgene into the
160
albumin locus . Although this approach was effective in rescuing lethality of CNS-I
mice, future improved strategies should be also considered for therapeutic applications.
Still, further progresses of the gene targeting technologies may be required to increase
their efficacy and to safely reach the clinics, avoiding important concerns such as the off-
target activity of the nucleases, the potential random insertion of the DNA carrying the
nucleases, long-term presence of the nucleases in the cell, and immune response against
the vehicle delivering the nucleases (i.e., the AAV vectors) and to the nucleases
themselves.
As mentioned above, AAV-mediated clinical trials for treating severe Crigler-Najjar
patients are ongoing, and the expected success of these approaches will open new
perspectives to patients, providing safe alternative therapies aimed to eliminate the risk of
bilirubin-induced brain damage, considerably improving their life quality.

Article Highlights
 The development of novel therapies for the Crigler-Najjar syndrome type I is
a clinical need. Phototherapy is effective temporarily and there is no
definitive cure except for liver transplantation, a procedure with many
shortcomings and risks.

 The availability of mouse models bearing Ugt1 null mutations with

phenotypes closely mimicking the human syndrome improved significantly
the understanding of the disease and paved the way to explore new
therapeutic approaches

 AAV-mediated gene therapy emerged as a valuable option for adult patients,

but still faces some limitations in pediatric and juvenile patients due to the
possibility of vector dilution in a growing liver.

 Immunomodulatory approaches blocking the generation of neutralizing anti-

AAV antibodies consequent to the first treatment are being tested. These

Page 22 of 45
 approaches will allow re-administration of the therapeutic AAV vector in the
case of loss of efficacy.

 Genome editing and targeted genome integration are very promising

alternative approaches that result in the permanent modification of the
genome, overcoming AAV vector dilution in pediatric subjects and ensuring
long-lasting correction of the genetic defect.

Funding
This paper was funded by Beneficentia Stiftung, Vaduz, Lichtenstein.

Declaration of Interests
A Muro has received ICGEB Intramural funds and financial support from Beneficentia
Stiftung. The authors have no other relevant affiliations or financial involvement with
any organization or entity with a financial interest in or financial conflict with the subject
matter or materials discussed in the manuscript apart from those disclosed.

Reviewer disclosures
Peer reviewers on this manuscript have no relevant financial relationships or otherwise to
disclose.

Page 23 of 45
Legends to the Figures

Figure 1. Bilirubin metabolism. A) The scheme depicts the chemical reactions required
to generate conjugated bilirubin. Enzymes, substrates and products are indicated; B)
Scheme showing the hepatic architecture, and transport of unconjugated bilirubin (UCB)
and conjugated bilirubin (CB) in the hepatocyte. The scheme of Panel B was modified
with permission from Frevert et al [173]

Figure 2. Hereditary unconjugated hyperbilirubinemias. The scheme depicts the

severity of hyperbilirubinemia in relation to UGT1A1 enzyme activity, and the treatments
applied for each condition. Gene therapy (in red) is an experimental approach, being
tested in clinical trials.

Figure 3. Therapeutic approaches for the Crigler-Najjar syndrome. The scheme

depicts the main bilirubin pathways, the established and potential treatments for the
Crigler-Najjar syndrome. Further details are described in Table 2.

Page 24 of 45
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Manuscript ID EOOD-2018-0042

"Advances in understanding disease mechanism and potential treatments for

Crigler-Najjar syndrome"

Table 1. Hereditary unconjugated hyperbilirubinemias.

Plasma Phototherapy Liver Liver damage

bilirubin levels treatment transplantation
Gilbert Syndrome Less than 3.5 None Not required No
mg/dl (60 µM)
Crigler-Najjar 3.5-20 mg/dl Limited According to No
syndrome type II (60-340 µM) phototherapy- the severity
Phenobarbital
Crigler-Najjar More than 20 Intensive Required In a low proportion of
syndrome type I mg/dl (340µM) phototherapy cases may present
(10-12 h/day) fibrosis 167, 168

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"Advances in understanding disease mechanism and potential treatments for

Crigler-Najjar syndrome"

Table 2 – Established and potential treatments for the Crigler-Najjar syndrome.

Treatment Application Advantages Disadvantages/risks References

Very invasive procedure; 5%
Liver Performed in CNS- mortality at 5 years after OLT; 24, 25, 81, 82
Permanent cure
Transplantation I and CNS-II organ rejection; life-long
immunosuppression
Long-term protection in Gunn
Hepatocyte Performed in CNS- 102-104, 106-109
Bridge therapy to OLT rats; poor engraftment and loss of
transplantation I and CNS-II
efficacy in patients
It is not a permanent cure; long
Routine treatment
Very efficient in the first years daily exposure times (10-14 65, 66, 70
Phototherapy for CNS-I and
of life h/day); loss of efficacy when the
CNS-II
patient grows; poor life quality
Increase of gene transcription
Routine treatment Has no or very minor effect in 69, 70
Phenobarbital and induction of residual
for CNS-II CNS-I patients; side effects
UGT1A1 activity
Agents modifying Capture UCB in the intestinal
enterohepatic lumen limiting reabsorption, or
Adverse effects such as diarrhea, 69, 96, 97, 171
circulation CNS-I and CNS-II enhance intestinal transit and
stomach cramps, etc.
(Orlistat, Calcium fecal elimination; may reach
Phosphate, etc) ~10% reduction in UCB
Increase plasma bilirubin-
binding capacity, reducing Monitoring of plasma bilirubin
Albumin Can be used in 36, 79, 93, 94
tissue bilirubin levels. levels does not reflect the risk of
administration acute situations
Frequently combined with neurological damage
exchange transfusion.
Not possible as long-term
Can be used in Reduce or block the production treatment. Some inhibitors 69, 70, 85, 87-89
HO-1 inhibitors
acute situations of bilirubin increase photosensitivity of the
skin
Minocycline Pre-clinical Can be used in acute situations Incompatible with phototherapy 59, 172
administration experimentation to reduce neurological damage treatments; side effects
Block the production of Patients and animal models with
Pre-clinical 86, 90, 91
BLVR inhibitors bilirubin; excess of BLV can be null mutations in the BLVR gene
experimentation
eliminated in urine and bile are compatible with life
Long-lasting in adults (FIX
Potential loss of efficacy in
clinical trials); moderately 114-118, 145, 149,
Clinical trials pediatric/juvenile patients;
Gene therapy invasive procedure; does not 150
ongoing adverse immune reactions; re-
preclude OLT in case of poor
administration not possible
efficiency or loss of efficacy
Permanent correction of the
genome; long-lasting; Off-target activity of nucleases;
Pre-clinical moderately invasive procedure; need of a different protocol for
Gene editing none
experimentation does not preclude OLT in case each disease-causing mutation;
of poor efficiency or loss of high viral titer required
efficacy
Permanent correction of the
genome; long-lasting; single
therapeutic approach for most
Pre-clinical disease-causing mutations; Off-target activity of nucleases; 159, 160
Gene targeting
experimentation moderately invasive procedure; high viral titer required
does not preclude OLT in case
of poor efficiency or loss of
efficacy

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"Advances in understanding disease mechanism and potential treatments for

Crigler-Najjar syndrome"

Table 3 – Clinical trials for Crigler-Najjar syndrome.

Clinicaltrials.gov NCT03223194 NCT03466463

identifier
Sponsor Audentes Therapeutics CureCN consortium
Vector AAV8 AAV8
Gene promoter TBG 1-AAT
Transgene Codon optimized UGT1A1 Codon optimized UGT1A1
Status 1st patient dosed Recruiting (expected dosing
2018)
Number of Cohorts 3 cohorts: 1.5E12 vg/kg; 6.0E12 2 cohorts; doses not reported
and doses vg/kg; and 1.5E13 vg/kg
Eligible ages 1 Year and older 9 Years and older
Administration route Intravenous, one dose Intravenous, one dose

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Figure 1

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Figure 2

Figure 3

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