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Adeno-associated viral gene therapy corrects a mouse model of argininosuccinic aciduria
Accepted Manuscript Adeno-associated viral gene therapy corrects a mouse model of argininosuccinic aciduria
Scott N. Ashley, Jayme M.L. Nordin, Jenny A. Greig, James M. Wilson PII: DOI: Reference:
S1096-7192(18)30195-1 doi:10.1016/j.ymgme.2018.08.013 YMGME 6399
To appear in:
Molecular Genetics and Metabolism
Received date: Revised date: Accepted date:
9 April 2018 26 August 2018 27 August 2018
Please cite this article as: Scott N. Ashley, Jayme M.L. Nordin, Jenny A. Greig, James M. Wilson , Adeno-associated viral gene therapy corrects a mouse model of argininosuccinic aciduria. Ymgme (2018), doi:10.1016/j.ymgme.2018.08.013
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ACCEPTED MANUSCRIPT Adeno-Associated Viral Gene Therapy Corrects a Mouse Model of Argininosuccinic Aciduria Scott N. Ashley, Jayme M. L. Nordin, Jenny A. Greig, and James M. Wilson*
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Gene Therapy Program, Department of Medicine, University of Pennsylvania, Philadelphia, PA,
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USA
*Correspondence should be addressed to J.M.W.:
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James M. Wilson, M.D., Ph.D.
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Gene Therapy Program Perelman School of Medicine
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University of Pennsylvania
Philadelphia, PA 19104, USA
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125 South 31st Street
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Phone: 215-898-0920; Fax: 215-494-5444
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E-mail: [email protected]
ACCEPTED MANUSCRIPT Abstract Argininosuccinic aciduria (ASA) is the second most common genetic disorder affecting the urea cycle. The disease is caused by deleterious mutations in the gene encoding argininosuccinate lyase (ASL); total loss of ASL activity results in severe neonatal onset of the disease, which is
The long-term complications of ASA, such as hypertension and neurocognitive
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and death.
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characterized by hyperammonemia within a few days of birth that can rapidly progress to coma
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deficits, appear to be resistant to the current treatment options of dietary restriction, arginine supplementation, and nitrogen scavenging drugs. Treatment-resistant disease is currently being
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managed by orthotopic liver transplant, which shows variable improvement and requires lifetime
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immunosuppression. Here, we developed a gene therapy strategy for ASA aimed at alleviating the symptoms associated with urea cycle disruption by providing stable expression of ASL We designed a codon-optimized human ASL gene packaged within an
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protein in the liver.
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adeno-associated virus serotype 8 (AAV8) as a vector for targeted delivery to the liver. To evaluate the therapeutic efficacy of this approach, we utilized a murine hypomorphic model of
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ASA. Neonatal administration of AAV8 via the temporal facial vein extended survival in ASA
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hypomorphic mice, although not to wild-type levels.
Intravenous injection into adolescent
hypomorphic mice led to increased survival and body weight and correction of metabolites
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associated with the disease.
Our results demonstrate that AAV8 gene therapy is a viable
approach for the treatment of ASA.
Highlights:
AAV gene therapy extends survival of adolescent ASA hypomorphic mice
AAV gene therapy extends survival of neonatal ASA hypomorphic mice
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ACCEPTED MANUSCRIPT
AAV gene therapy normalizes plasma argininosuccinic acid and citrulline levels in ASA hypomorphic mice
AAV gene therapy normalizes serum transaminase levels in ASA hypomorphic mice
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Keywords: AAV, Gene Therapy, Argininosuccinic Aciduria, Liver, Urea Cycle
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Abbreviations: Argininosuccinic aciduria (ASA); argininosuccinate lyase (ASL); adeno-
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associated virus (AAV); thyroxine-binding globulin (TBG); bovine growth hormone (bGH);
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genome copies (GC); intravenous (IV); analysis of variance (ANOVA); immunohistochemistry
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(IHC); alanine aminotransferase (ALT); aspartate aminotransferase (AST)
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ACCEPTED MANUSCRIPT 1. Introduction Argininosuccinic aciduria (ASA) is an autosomal recessive disorder of the urea cycle caused by mutations within the argininosuccinate lyase (ASL) gene that result in impaired arginine synthesis. Newborn screening programs in the United States are important for early detection of
diagnosis
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determined by detection of the unique metabolite marker
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differential
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ureagenesis disorders; however, due to the similarity of ASA to other urea cycle disorders,
Neonatal
ASA, characterized by
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primary manifestations: neonatal and late onset.
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argininosuccinic acid by tandem mass spectrometry in patient plasma [1, 2]. The disease has two
hyperammonemia within days following birth, can be treated by hemodialysis followed by
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lifetime maintenance care to reduce the risk of further episodes [3-5]. Late-onset ASA has a less
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severe phenotype that includes episodic hyperammonemia triggered by acute infection or stress , as well as neurocognitive impairment with associated learning or behavioral abnormalities [4].
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Patients with ASA can also manifest symptoms unrelated to hyperammonemia due to the lack of
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nitric oxide production in both forms of the disorder, including neurocognitive deficiencies, cirrhosis of the liver, and systemic hypertension [4-7].
Standard maintenance care includes
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protein restriction, arginine supplementation, and nitrogen scavenging drugs (e.g., sodium
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benzoate, sodium phenylburyrate, and glycerolphenylburyrate) [8, 9]. Individuals with recurrent hyperammonemia or cirrhosis of the liver can also undergo liver transplant as a diseasemodifying process. However, lifetime immunosuppression is required following transplant and early transplantation cases showed lower levels of improvement compared to other urea cycle disorders, although more recent case studies showed positive health and quality of life outcomes [10-14].
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ACCEPTED MANUSCRIPT As the absence of ASL from hepatocytes causes ASA-related hyperammonemia [12, 15, 16], liver-targeted gene therapy has the potential to benefit patients with severe disease. Adenoassociated virus (AAV)-mediated gene therapy has demonstrated both therapeutic efficacy and low immunogenicity in murine models for the treatment of other urea cycle disorders (e.g.,
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ornithine transcarbamylase deficiency, arginase deficiency, and citrullinemia) [17-23].
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However, ASA presents a unique challenge as the enzyme is essential not only for the removal
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of waste nitrogen via the urea cycle, but also for the clearance of argininosuccinic acid through its conversion to arginine. The buildup of argininosuccinic acid is thought to cause some of the
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unique symptoms of the disease, including hepatic and cognitive deficits [4, 5]. Based on results
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from patients with ASA who have received liver transplants [15, 16, 24], liver-targeted gene therapy is uniquely positioned to restore the urea cycle and increase quality of life without the
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need for a highly invasive procedure or a continued drug regimen. In this study, we developed a
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gene therapy for ASA using an AAV8 vector expressing the human ASL gene under the control of the liver-specific thyroxine-binding globulin (TBG) promoter, and examined therapeutic
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efficacy in both neonatal and adolescent ASA hypomorphic mice.
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ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1. Mice ASA hypomorphic mice on a C57BL/6 background were acquired from The Jackson Laboratory (Bar Harbor, ME) and a colony was maintained at the University of Pennsylvania under specific All animal procedures and protocols were approved by the
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pathogen-free conditions.
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Institutional Animal Care and Use Committee of the University of Pennsylvania.
2.2. Vectors
promoter
with
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(AAV8.TBG.hASLco.bGH).
bovine
growth
hormone
(bGH)
polyA
signal
sequence
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TBG
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AAV8 vectors expressing human codon-optimized ASL were designed under the control of a
AAV vectors were produced by the Penn Vector Core at the
2.3. Neonatal gene therapy
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University of Pennsylvania, as previously described [25].
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During the first 24 h following birth, ASA hypomorphic pups (n=4 low dose, n=7 high dose)
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received intravenous (IV) administration of 1010 or 1011 genome copies (GC)/mouse of AAV8.TBG.hASLco.bGH via the temporal facial vein.
Mice were weighed throughout the
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study and genotyped upon weaning. Mice were sacrificed upon reaching median survival, and tissues were collected for histology.
2.4. Adolescent gene therapy ASA hypomorphic mice four to five weeks of age received IV administration of 1013 or 6x1013 GC/kg of AAV8.TBG.hASLco.bGH via the retro orbital vein (n=8).
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Mice were weighed
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Sub-mandibular bleeds were performed weekly to collect plasma for
monitoring liver transaminases and urea cycle metabolites.
2.5. Serum analyses
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Plasma samples were submitted to Antech Diagnostics (Irvine, CA) for analysis of liver
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transaminases and Charles River Laboratories (Worcester, MA) for analysis of arginine,
acetonitrile.
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citrulline, and argininosuccinic acid by LC-MS/MS following protein precipitation with Mice were sacrificed after three months and tissues were collected for
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biodistribution and histology.
2.6. ASL immunohistochemistry
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Tissues were fixed in formalin for a minimum of 24 h and then paraffin embedded. Sections
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were deparaffinized through an ethanol and xylene series, boiled for 6 min in 10 mM citrate buffer (pH 6.0) for antigen retrieval, and sequentially treated with 2% H 2O2 (15 min),
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avidin/biotin blocking reagents (15 min each; Vector Laboratories, Burlingame, CA), and Sections were then
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blocking buffer (10 min; 1% donkey serum in PBS + 0.2% Triton).
incubated with a rabbit serum against ASL (1 h; HPA016646, Sigma-Aldrich, St. Louis, MO)
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and biotinylated secondary anti-rabbit antibodies (45 min; Jackson ImmunoResearch, Westgrove, PA) diluted in blocking buffer at the manufacturer’s recommended concentration. A Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA) was used according to the manufacturer's instructions with 3,3'-diaminobenzidine as the substrate to stain bound antibodies.
2.7. Sirius Red and HE
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ACCEPTED MANUSCRIPT To detect liver fibrosis, Sirius red staining was performed on paraffin sections. Deparaffinized sections were stained for 90 min in a solution containing 0.1% (w/v) Direct Red (Sigma) and 4% (w/v) picric acid (Sigma-Aldrich, St. Louis, MO), and then washed with 0.01 N HCl (2 x 1 min), dehydrated through an ethanol and xylene series, and coverslipped. Paraffin sections were
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stained with hematoxylin and eosin (H&E) according to standard protocols.
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2.8. Biodistribution
Liver samples were frozen on dry ice at the time of necropsy, and DNA was extracted using the
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QIAamp DNA Mini Kit (Qiagen, Valencia, CA). Detection and quantification of vector GCs in
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extracted DNA were performed by real-time polymerase chain reaction, as previously described [26]. Briefly, genomic DNA was isolated, and vector GCs were quantified using primers/probes
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designed against the promoter sequence of the vector. Quantification of GCs from liver was
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2.9. ASL activity assay
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performed on one liver sample from each mouse.
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Liver (25-30 mg) was added to 200 µl of cold homogenizing buffer containing 50 mM phosphate buffer (pH 7.5) and proteinase inhibitors (EDTA-free proteinase inhibitor cocktail; Roche, Basel,
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Switzerland), and homogenized using Tissue Lyser II (Qiagen, Valencia, CA), at a frequency of 30 for 30 seconds.
Homogenates were centrifuged at 10,000 x g for 20 min at 4ºC, and
supernatants were kept frozen at 80ºC. Lysate (2 µl) was added to 48 µl of 50 mM phosphate buffer (pH 7.3), 3.6 mM argininosuccinic acid (Sigma Aldrich, St. Louis, MO). The reaction was incubated at 37ºC for 1 h and stopped by heating at 80ºC for 20 min. Fumarate was measured with Fumarate Assay Kit (Sigma Aldrich, St. Louis, MO) per the manufacturer’s
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ACCEPTED MANUSCRIPT specifications using 5 µl of reaction sample mixture.
2.10. Statistical analysis Comparison of survival was performed by logrank test with each experimental group compared
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to uninjected control groups, unless otherwise stated. Other parameters were compared by one-
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way analysis of variance (ANOVA) with Dunnett’s multiple comparison test comparing each
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group to the untreated wild-type control group, unless otherwise stated. *p < 0.05, **p < 0.01,
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***p < 0.001.
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3. Results
3.1. An ASA hypomorphic mouse model enables the study of both neonatal and adolescent mice
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To evaluate our gene therapy approach, we first considered which of the two ASA mouse models
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to use for our studies. A knockout model (Asl -/-) was created by replacing exons 8 and 9 with a 1,400 bp neomycin cassette, resulting in a frame shift in the mRNA beginning with exon 10 [27].
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All Asl-/- mice have elevated plasma ammonia, argininosuccinic acid, and citrulline, as well as
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low plasma arginine. However, these mice die within 48 hours of birth, so this model can only be used to study treatment of a neonatal cohort. As an alternative, an ASA hypomorphic mouse
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model has been developed, where a 1,200 bp neomycin cassette was inserted into intron 9, resulting in reduced, but not ablated, mRNA levels and slightly prolonged survival [6]. These mice have the same characteristic variations in amino acids and liver metabolites, and they display a sparse fur coat. Therefore, for the current study we used the ASA hypomorphic mouse. We confirmed that these mice have a mean survival of 22 days (Figure 1A). Homozygous ASA hypomorphic pups were indistinguishable from heterozygous or wild-type littermates until five
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ACCEPTED MANUSCRIPT days after birth, at which time the hypomorphic mice failed to develop a fur coat and were visibly smaller.
3.2. Prophylactic AAV8 gene therapy extends survival of neonatal ASA hypomorphic mice
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We next evaluated AAV8 therapy in neonatal mice. We performed IV injections via the temporal
We genotyped mice after weaning and monitored the vector-administered
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24 hours of birth.
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facial vein with 1010 or 1011 GC/mouse of the AAV8.TBG.hASLco.bGH vector within the first
ASA hypomorphic mice for survival. Vector administration at both doses significantly increased
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survival compared to untreated ASA hypomorphic mice (p < 0.01; Figure 1B). The increase in
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median survival was dose dependent, with a median survival of 165 days for the 1011 GC/mouse group compared to 136 days for the 1010 GC/mouse group. Weight gain for vector-administered
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ASA hypomorphic mice was comparable to wild-type littermates throughout the study, with the
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exception of the female vector-administered hypomorphic mice at the last time point evaluated
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(day 63; Figures 1C).
We used
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Once the mice reached median survival, we euthanized and necropsied them.
immunohistochemistry (IHC) of the liver to visualize ASL protein expression (Figure 2). IHC
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revealed a small number of strongly stained positive cells. The low level of transgene expression observed in these adolescent mice was expected, as the rapid proliferation of hepatocytes during early development has been shown to significantly dilute non-integrating vector genomes [2830].
Despite the low level of transgene expression observed at the time of necropsy, ASL
activity was sufficient to allow all vector-treated ASA hypomorphic mice to live beyond the 22 day mean survival of untreated ASA hypomorphic mice. However, as expected without re-
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ACCEPTED MANUSCRIPT administration, we confirmed that vector dilution as the animal grew led to reduced efficacy and resulted in premature death (Figure 1B).
3.3. AAV8 gene therapy treatment extends survival, increases weight, and normalizes serum
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transaminase levels in adolescent ASA hypomorphic mice
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In addition to a prophylactic gene therapy model following neonatal administration, we
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investigated the potential treatment effects of AAV8 gene therapy in adolescent ASA hypomorphic mice. We administered AAV8 to 30-day-old adolescent mice via the retro orbital
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vein; the standard adolescent mouse IV administration route via the tail vein could not be used
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for these adolescent ASA hypomorphic mice as their average weight was 8.8 g. We collected plasma from the first cohort of female mice for baseline metabolite and transaminase evaluation
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prior to IV administration with 6x1013 GC/kg of vector.
Mice that had been bled had high
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mortality rates prior to or shortly after vector administration, likely due to reduced blood volume and were omitted from the survival data set for this cohort (Figure 3A). Therefore, we did not
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determine baseline values for the other cohorts. Further cohorts of adolescent ASA hypomorphic
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mice were either untreated or administered IV with 1013 GC/kg of vector. Survival of mice in all vector-administered cohorts increased compared to untreated ASA hypomorphic mice (p <
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0.001; Figure 3A). Mean survival was extended to 91 days in the low-dose female group, with the study terminated before the high-dose female and low-dose male cohorts had lost enough mice to determine mean survival (Figure 3A). We observed a sex difference in survival, with untreated female ASA hypomorphic mice having increased mean survival compared to untreated male ASA hypomorphic mice (p < 0.001). However, following low-dose vector administration, male mice showed increased survival and weight gain compared to female mice, which could
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ACCEPTED MANUSCRIPT possibly be due to an androgen-dependent, species-specific effect on AAV transduction of the liver as previously described [31]. We observed a dose-dependent increase in weight gain; however, even mice administered 6x1013 GC/kg of vector did not match wild-type body weights (Figure 3B). All vector-treated groups had reductions in plasma alanine aminotransferase (ALT)
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and aspartate aminotransferase (AST) compared to untreated controls (Figure 3C and 3D).
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3.4. AAV gene therapy normalizes plasma argininosuccinic acid and citrulline levels in ASA
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hypomorphic mice
AAV gene therapy has the unique potential to restore the urea cycle in hepatocytes without the Therefore, we evaluated the effect of our AAV8 vector on
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need for a liver transplant.
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metabolites associated with ASA and other aspects of the urea cycle. Argininosuccinic acid is broken down into arginine and fumarate by the ASL enzyme, leading to elevations in
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argininosuccinic acid and citrulline (a substrate of the reaction upstream of ASL), and reductions
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in arginine.
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Plasma citrulline was elevated to 474 ± 155 µmol in the hypomorphic mice compared to 58.3 ± In the high-dose female and low-dose male
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14.4 µmol in the wild-type mice (Figure 4A).
groups, we saw a dramatic reduction of citrulline in plasma, resulting in no significant difference
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in citrulline levels compared to wild-type littermates indicating correction of the metabolite (Figure 4A). We did not observe an effect of the low dose of vector on plasma citrulline levels in the female mice. Plasma argininosuccinic acid, which is the metabolite that differentiates ASA from other urea cycle disorders and is thought to play a role in the unique symptoms of the disease, was elevated from 163.6 ± 60.0 µmol in the hypomorphic mice compared to 1.53 ± 1.13 µmol in the wild-type mice (Figure 4B). Plasma argininosuccinic acid was corrected to normal
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ACCEPTED MANUSCRIPT levels in the high-dose female group (Figure 4B), but remained elevated in both the male and female low-dose cohorts, indicating a steep dose effect.
Wild-type mice had plasma arginine levels in the range of 45-185 µM with a mean of 95 µM Characterization of arginine levels in the ASA hypomorphic mouse was
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(Figure 4C).
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complicated because of less dramatic differences between values in normal and ASA
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hypomorphic mice, and the early mortality of the mice in the absence of treatment. In fact, at the initiation of the study ASA hypomorphic mice had arginine levels similar to the wild type (75
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µM) (Figure 4C). However, at day 35 we observed significant reductions in arginine levels from
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wild-type mice compared to male and female hypomorphic cohorts treated with 1013 GC/kg of AAV8 vector, suggesting incomplete correction. However, the extent of the therapeutic affect is
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unclear as we do not have samples from untreated hypomorphic mice at this age group to analyze
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(Figure 4C). Incomplete correction may also be due to the liver-restricted nature of our therapy,
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synthesis in the body.
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where to correct arginine we would want to target to the kidney, the primary site of arginine
Upon termination of the study, we sacrificed the mice and harvested liver for determination of
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ASL protein distribution, liver disease progression, and vector GCs. Female ASA hypomorphic mice administered with the high dose of vector had a strong presence of ASL protein in the liver as measured by IHC (Figure 5A). Male and female mice dosed with the low dose showed similar levels of staining.
Activity of ASL protein in liver lysate of high-dose females as
measured by an activity assay was on average 25% of wild-type levels and statistically significantly higher than untreated hypomorphic mice, which had a mean activity of 3% (Figure
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ACCEPTED MANUSCRIPT 5B). The activity in mice administered with the low vector dose was not statistically higher than in the untreated hypomorphic mice.
Vector genome copy analysis on liver samples demonstrated that ~four-fold higher vector GCs
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were present in hepatocytes in the high-dose cohort compared to the low-dose groups (Figure
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5C). These results demonstrate that with sufficient transduction of liver by an ASL gene therapy
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vector, survival in the ASA hypomorphic mouse model can be greatly extended.
This is
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correction of metabolite markers to normal levels.
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concurrent with a reduction in liver transaminases indicating reduced hepatic toxicity and the
The primary histopathologic findings in the liver of ASA hypomorph mice that received either
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low- or high-dose vector included variable hepatocellular changes consisting of enlargement of
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both the nucleus and cytoplasm (karyocytomegaly) with and without individual hepatocellular necrosis and regeneration (indicated by increased incidence of mitotic figures). The more severe
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cases exhibited hepatocellular dissociation with and without lobular collapse. Other findings
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included mixed cellular infiltrates, fibrosis, and higher scores of bile duct and oval cell hyperplasia indicating a more severe phenotype (Figure 6). H&E staining revealed that low-dose
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male mice (Figure 7A and 8A) appeared to have more severe histopathological events than the equivalently dosed female cohort (Figure 7B); by contrast, the high-dose female group trended better than their low-dose counterpart (Figure 7C and 7D).
We observed minimal
histopathologic findings in untreated ASA hypomorphic mice compared to dosed cohorts (Figure 7E). We believe this result is due to the early death of these untreated mice (~ study day 15), as all of the histology of dosed groups were generated from samples taken at the termination of the
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ACCEPTED MANUSCRIPT study (study day 98). In addition, we independently scored the liver fibrosis based on location and amount of hepatic infiltration on sections using Sirius red. Although we observed a range of scores for both doses, animals from the low-dose male cohort group (Figure 8B) more commonly showed the presence of and higher scores for fibrosis, compared to the low-dose (Figure 8C) or Again, untreated mice did not develop observable
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histopathology due to the limited time for it to develop (Figure 8E).
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high-dose (Figure 8D) female cohorts.
4. Discussion
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ASA, caused by a deficiency in ASL activity, is characterized by the dysregulation of the urea
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cycle that inhibits arginine synthesis and nitric oxide production [4]. Here, we showed that delivery of the ASL gene into hepatocytes increased survival and weight gain of ASA
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hypomorphic mice. Median survival for ASA hypomorphic mice was 22 days post birth due to a
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failure to thrive marked by an increase in liver transaminases and elevations in urea cycle metabolites. We were able to increase survival of these mice in a dose-dependent manner via
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neonatal administration of vector through the facial vein. However, treated mice did not gain
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weight to an equivalent degree as their wild-type littermates; liver histology suggested that dilution of vector genomes occurred with growth of the animal, which obscures regional
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transduction differences, consistent with what has been previously reported [28-30]. We believe that this vector dilution contributed to the early death of treated hypomorphic mice, where ASL expression necessary for survival may have dropped lower than a critical threshold resulting in a buildup of toxic metabolites. This will be a critically important phenomenon in advancing gene therapy for infant patients with ASA, as readministration of gene therapy may be necessary. Successful readministration of vector to the liver can prove difficult due to circulating
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ACCEPTED MANUSCRIPT neutralizing antibodies to the vector capsid that would develop after the initial administration. However, recent advances in immune suppression have shown potential for either preventing AAV antibody development or reducing the circulating antibodies to other AAV serotypes,
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allowing for repeat dosing [32, 33].
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To determine the efficacy of ASA gene therapy in more developed livers, we administered
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vector via a retro orbital IV injection to adolescent mice. These mice exhibited both increased survival and weight gain, although only the high dose resulted in survival similar to wild-type
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littermates. One potential cause of the continued early morbidity in treated adolescent mice may
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be the vector genome dilution, because although these mice are older than the newborn treatment groups, they have not yet reached full size. Indeed, based on our monitoring of weight of
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untreated ASA hypomorphic mice, a successful start to the therapy may ultimately result in an
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attenuation of its efficacy as it allows the treated mice to increase in weight rapidly.
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Cirrhosis of the liver is one of the hallmarks of ASA, affecting individuals more commonly than
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other urea cycle disorders, and is a primary reason that patients undergo liver transplant [3]. We did not observe liver pathology in untreated ASA hypomorphic mice following review of liver
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sections by a board-certified veterinary pathologist potentially because the affected mice died before cirrhosis could develop. However, we did observe extensive histopathology in some treated groups, especially the low-dose male cohort.
The low-dose female cohort had a
comparatively less severe phenotype that was further reduced in the high-dose cohort. These results could be indicative of a sex bias in treatment efficacy toward more correction in female mice; however, based on metabolite analysis of citrulline we might predict that the opposite is
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ACCEPTED MANUSCRIPT occurring. Another possibility is that ASA liver pathology is more severe in male mice. This line of inquiry requires further understanding of the natural history of the ASA hypomorph, as no reports in the literature suggest a sex difference in the severity of ASA liver cirrhosis. Overall, the severity of histopathological findings in the female cohorts did seem to improve in a dose-
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dependent manner indicating that gene therapy could be effective in treating one of the primary
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reasons patients with ASA require liver transplant [3]. We also observed reduced transaminase
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levels in all treated mouse groups at most time points studied. Furthermore, mice in the highdose group showed a trend for corrected arginine levels, indicating that AAV gene therapy might
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correct arginine deficiency, which is not corrected sufficiently with liver transplant [3, 4]. The
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high-dose group also achieved normalization of plasma argininosuccinic acid. This encouraging
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result suggests that argininosuccinic acid is being cleared from the liver.
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Citrulline, a metabolite upstream of ASL, was normalized in both high-dose administered females and low-dose administered males.
Evidence suggests that an androgen-dependent
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mechanism enhances transduction in male mice (27). However, we did not observe a difference
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in the vector genome copy number between low-dose groups based on sex, suggesting a potential
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difference in disease severity based on sex [31].
One of the major difficulties facing gene therapy treatment of ASA is the importance of ASL in non-hepatic metabolic pathways outside of the urea cycle and how they may negatively impact therapeutic efficacy [13, 14]. ASL is required for both cleavage of argininosuccinic acid into arginine in the kidneys, providing a major of source of serum arginine [34], and production of nitric oxide by endothelial cells lining the arteries [35]. Helper-dependent adenoviral vector
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ACCEPTED MANUSCRIPT gene therapy for this hypomorphic mouse model of ASA showed similar improvements in repair of the urea cycle compared to our vector, but was unable to overcome other disease phenotypes associated with non-liver expression of ASL; we believe this would not be different for our gene therapy approach [36]. Initial longitudinal studies of orthotopic liver transplants for patients with
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ASA, whose outcome a liver-targeted gene therapy could mimic, displayed a less positive
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outcome compared to other urea cycle disorders [14]. A debate has existed among researchers as
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to whether this low efficacy is due to insufficient arginine, loss of nitric oxide synthesis contributing to the cirrhosis of the liver, or buildup of argininosuccinic acid [12, 37]. However,
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more recent studies of liver transplants have shown stronger positive outcomes [12, 15, 16].
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Taken together, it would seem prudent to consider continuing arginine supplementation after gene therapy, and with early treatment and improved care outcomes it may come to resemble
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more recent transplant success.
5. Conclusions
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In summary, we have demonstrated the feasibility of gene therapy for ASA using AAV8 vector
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technology. However, both routes of administration (IV via the temporal facial vein in newborns and retro orbitally in adolescent mice) pose some challenges. Neonatal administration was able
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to extend survival compared to untreated ASA hypomorphic mice, but not to the full lifespan of wild-type mice. This could be solved with readministration of the gene therapy vector at a later time point, an important consideration for future development.
Adolescent vector
administration, while able to correct metabolites at the highest dose tested, was less efficacious at a lower dose.
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ACCEPTED MANUSCRIPT Acknowledgments We thank Deirdre McMenamin, Christine Draper, Hongwei Yu, and Meardey So for invaluable technical assistance.
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Disclosures
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J.M. Wilson is an advisor to, holds equity in, and has a sponsored research agreement with
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REGENXBIO and Scout Bio; he also has a sponsored research agreement with Ultragenyx, Biogen, and Janssen, which are licensees of Penn technology. In addition, he has sponsored
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research agreements with Precision Biosciences and Moderna Therapeutics.
J.M.W. is an
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inventor on patents that have been licensed to various biopharmaceutical companies.
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Funding Source
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This study was funded by internal funding from the University of Pennsylvania.
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Martinelli, P.S. Crespo, R. Santer, A. Servais, V. Valayannopoulos, M. Lindner, V. Rubio, C.
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Dionisi-Vici, Suggested guidelines for the diagnosis and management of urea cycle disorders
[3]
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Orphanet journal of rare diseases 7 (2012) 32.
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ACCEPTED MANUSCRIPT Figure Legends
Figure 1. AAV8 gene therapy extends survival of an ASA hypomorphic mouse model. (A) A survival curve of ASA hypomorphic mice is shown (n = 81). (B) ASA hypomorphic mice
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were injected IV via the temporal vein with 10 10 GC/mouse (n=5) or 1011 GC/mouse (n=6) of
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AAV8.TBG.hASLco and monitored for survival. Comparisons to uninjected ASA hypomorphic
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mice (solid line) are shown. The weight of female (C) and male (D) ASA hypomorphic mice administered with 1011 GC/mouse of AAV8.TBG.hASLco was monitored, and compared to
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wild-type (WT) mice and ASA hypomorphic mice that did not receive gene therapy. Error bars
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= standard error of the mean (SEM); **p < 0.01, ***p <0.001.
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Figure 2. ASL protein expression in the liver of newborn injected mice.
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ASA hypomorphic mice were injected IV via the temporal vein with 10 10 GC/mouse (n=5) or 1011 GC/mouse (n=6) of AAV8.TBG.hASLco.
Mice were necropsied after cohort-specific
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median survival was reached. Livers were harvested and IHC was performed for detection of the
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ASL protein, and compared to wild-type (WT), heterozygous (Het), and untreated ASA hypomorphic mice.
Central veins are denoted by asterisks.
Representative sections from
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different mice in each cohort are shown.
Figure 3.
Sex difference in survival and efficacy of AAV8 gene therapy in ASA
hypomorphic mice. ASA hypomorphic mice were injected IV via the retro orbital vein on P30 with 1013 or 6x1013 GC/kg of AAV8.TBG.hASLco (n=8/group). Mice were monitored for survival (A) and weight
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ACCEPTED MANUSCRIPT (B). Comparisons to uninjected ASA hypomorphic mice are shown (female, n=3; male, n=4). Mice were bled and plasma was analyzed for the liver transaminases AST (C) and ALT (D). Comparison of values to uninjected ASA hypomorphic mice (no vector) at the same time point
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are shown (n=5). Error bars = SEM; **p < 0.01, ***p <0.001.
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Figure 4. High-dose AAV8 gene therapy corrects liver metabolism in ASA hypomorphic
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mice.
ASA hypomorphic mice were injected IV via the orbital vein on P30 with 1013 or 6x1013 GC/kg
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of AAV8.TBG.hASLco (n=8/group). Mice were bled and plasma was analyzed for components
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of the urea cycle, including (A) citrulline, (B) ASA, and (C) arginine. Comparisons to levels in wild-type (WT) mice are shown (n=22), ASA hypomorph control (n=12). Error bars = SEM; ns,
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not significant; *p < 0.05, **p < 0.01, ***p <0.001.
Figure 5. Detection of ASL protein expression and genome copies in the liver of adolescent
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injected mice.
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ASA hypomorphic mice were injected IV via the retro orbital vein at P30 with 1013 (n=8/sex) or 6x1013 GC/kg of AAV8.TBG.hASLco (n=8/group). (A) Livers were harvested and IHC was
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performed for detection of the ASL protein. Each section is a representative sample from a different mouse within each cohort.
Central veins are denoted by asterisks.
(B) Liver
homogenates were assayed for ASL activity compared to wild-type levels (data presented as percentage of wild-type activity). Analysis of activity was performed by a one-way ANOVA with Dunnett’s multiple comparison test comparing each group to uninjected ASA hypomorphic mice (no vector). (C) Following extraction of DNA from liver, vector GCs were determined.
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ACCEPTED MANUSCRIPT Error bars = SEM; ***p < 0.001.
Figure 6.
Histopathologic findings in the liver of a mouse model of ASL administered
either a low (1x1013 GC) or high dose (6x1013 GC) of AAV.
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ASA hypomorphic mice were injected IV via the retro orbital vein at P30 with 10 13 (n=8/sex) or
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6x1013 GC/kg of AAV8.TBG.hASLco (n=8/group). Livers were harvested and H&E and Sirius
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red staining were performed for observation of histopathology. Findings from each category were ranked from 0-5 for hepatocellular changes with 0 being no finding, 5 being marked
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karyocytomegaly (>90% of hepatocytes within a lobule) with lobular collapse and numerous
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single-cell necrosis, and 1-3 denoting increasing severity.
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Figure 7. Histopathologic findings in the liver of a mouse model of ASL administered with
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either a low (1x1013 GC) or high dose (6x1013 GC) of AAV. The images depict representative trends of the histopathological findings for each dose group.
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(A) A male mouse received a low dose of vector and exhibited grade 4 hepatocellular changes
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characterized by severe karyocytomegaly (51-90% of hepatocytes within a lobule) with extensive hepatocellular dissociation and frequent individual cell necrosis; however, generally
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normal hepatocellular architecture was maintained. This animal also had oval cell (grade 3) and bile duct (grade 1) hyperplasia.
(B) A female mouse also received a low dose of vector;
however, hepatocellular changes were less severe (grade 2) characterized by mild karyocytomegaly (< 10% of hepatocytes within a lobule) with normal hepatic architecture and no individual cell necrosis.
This animal had no oval or bile duct hyperplasia.
(C) The
histopathologic findings were similar in a female mouse administered with a high dose of vector.
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ACCEPTED MANUSCRIPT (D) However, other animals in this dose group had no significant liver findings, as depicted in the displayed mouse. H&E, Scale bar = 100 µm.
Figure 8.
Variable fibrosis was observed in the liver of a mouse model of ASL
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administered with either a low (1x1013 GC) or high dose (6x1013 GC) of AAV.
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Sirius red- and H&E-stained liver sections were scored based on location and amount of hepatic
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infiltration. The severe cases (grade 3) were mostly in the low-dose group and were clearly visible on H&E staining. (A) In a male mouse from the low-dose cohort, the liver had regions of
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bridging fibrosis between portal and occasionally centrilobular areas with architectural disruption
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and individualization of hepatocytes. The presence of hepatocellular mitotic figures, indicative of regeneration, was frequently observed in these areas (inset, scale bar = 20 pixel). In these
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cases, the fibrosis was frequently accompanied by oval cell and bile duct hyperplasia. Sirius red
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staining highlighted the fibrosis in less severely affected sections. (B) A male mouse in the lowdose group exhibited regional bridging portal fibrosis (grade 2) with no architectural distortion,
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(C) whereas only periportal fibrosis (grade 1) was observed in a female mouse from the high-
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dose group. (D) No fibrosis was observed in another female mouse from the high-dose group.
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Sirius red, Scale bar = 100 µm.
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Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Scott N. Ashley, Jayme M.L. Nordin, Jenny A. Greig, James M. Wilson PII: DOI: Reference:
S1096-7192(18)30195-1 doi:10.1016/j.ymgme.2018.08.013 YMGME 6399
To appear in:
Molecular Genetics and Metabolism
Received date: Revised date: Accepted date:
9 April 2018 26 August 2018 27 August 2018
Please cite this article as: Scott N. Ashley, Jayme M.L. Nordin, Jenny A. Greig, James M. Wilson , Adeno-associated viral gene therapy corrects a mouse model of argininosuccinic aciduria. Ymgme (2018), doi:10.1016/j.ymgme.2018.08.013
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ACCEPTED MANUSCRIPT Adeno-Associated Viral Gene Therapy Corrects a Mouse Model of Argininosuccinic Aciduria Scott N. Ashley, Jayme M. L. Nordin, Jenny A. Greig, and James M. Wilson*
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Gene Therapy Program, Department of Medicine, University of Pennsylvania, Philadelphia, PA,
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USA
*Correspondence should be addressed to J.M.W.:
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James M. Wilson, M.D., Ph.D.
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Gene Therapy Program Perelman School of Medicine
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University of Pennsylvania
Philadelphia, PA 19104, USA
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125 South 31st Street
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Phone: 215-898-0920; Fax: 215-494-5444
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E-mail: [email protected]
ACCEPTED MANUSCRIPT Abstract Argininosuccinic aciduria (ASA) is the second most common genetic disorder affecting the urea cycle. The disease is caused by deleterious mutations in the gene encoding argininosuccinate lyase (ASL); total loss of ASL activity results in severe neonatal onset of the disease, which is
The long-term complications of ASA, such as hypertension and neurocognitive
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and death.
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characterized by hyperammonemia within a few days of birth that can rapidly progress to coma
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deficits, appear to be resistant to the current treatment options of dietary restriction, arginine supplementation, and nitrogen scavenging drugs. Treatment-resistant disease is currently being
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managed by orthotopic liver transplant, which shows variable improvement and requires lifetime
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immunosuppression. Here, we developed a gene therapy strategy for ASA aimed at alleviating the symptoms associated with urea cycle disruption by providing stable expression of ASL We designed a codon-optimized human ASL gene packaged within an
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protein in the liver.
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adeno-associated virus serotype 8 (AAV8) as a vector for targeted delivery to the liver. To evaluate the therapeutic efficacy of this approach, we utilized a murine hypomorphic model of
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ASA. Neonatal administration of AAV8 via the temporal facial vein extended survival in ASA
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hypomorphic mice, although not to wild-type levels.
Intravenous injection into adolescent
hypomorphic mice led to increased survival and body weight and correction of metabolites
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associated with the disease.
Our results demonstrate that AAV8 gene therapy is a viable
approach for the treatment of ASA.
Highlights:
AAV gene therapy extends survival of adolescent ASA hypomorphic mice
AAV gene therapy extends survival of neonatal ASA hypomorphic mice
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ACCEPTED MANUSCRIPT
AAV gene therapy normalizes plasma argininosuccinic acid and citrulline levels in ASA hypomorphic mice
AAV gene therapy normalizes serum transaminase levels in ASA hypomorphic mice
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Keywords: AAV, Gene Therapy, Argininosuccinic Aciduria, Liver, Urea Cycle
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Abbreviations: Argininosuccinic aciduria (ASA); argininosuccinate lyase (ASL); adeno-
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associated virus (AAV); thyroxine-binding globulin (TBG); bovine growth hormone (bGH);
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genome copies (GC); intravenous (IV); analysis of variance (ANOVA); immunohistochemistry
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(IHC); alanine aminotransferase (ALT); aspartate aminotransferase (AST)
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ACCEPTED MANUSCRIPT 1. Introduction Argininosuccinic aciduria (ASA) is an autosomal recessive disorder of the urea cycle caused by mutations within the argininosuccinate lyase (ASL) gene that result in impaired arginine synthesis. Newborn screening programs in the United States are important for early detection of
diagnosis
is
determined by detection of the unique metabolite marker
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differential
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ureagenesis disorders; however, due to the similarity of ASA to other urea cycle disorders,
Neonatal
ASA, characterized by
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primary manifestations: neonatal and late onset.
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argininosuccinic acid by tandem mass spectrometry in patient plasma [1, 2]. The disease has two
hyperammonemia within days following birth, can be treated by hemodialysis followed by
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lifetime maintenance care to reduce the risk of further episodes [3-5]. Late-onset ASA has a less
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severe phenotype that includes episodic hyperammonemia triggered by acute infection or stress , as well as neurocognitive impairment with associated learning or behavioral abnormalities [4].
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Patients with ASA can also manifest symptoms unrelated to hyperammonemia due to the lack of
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nitric oxide production in both forms of the disorder, including neurocognitive deficiencies, cirrhosis of the liver, and systemic hypertension [4-7].
Standard maintenance care includes
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protein restriction, arginine supplementation, and nitrogen scavenging drugs (e.g., sodium
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benzoate, sodium phenylburyrate, and glycerolphenylburyrate) [8, 9]. Individuals with recurrent hyperammonemia or cirrhosis of the liver can also undergo liver transplant as a diseasemodifying process. However, lifetime immunosuppression is required following transplant and early transplantation cases showed lower levels of improvement compared to other urea cycle disorders, although more recent case studies showed positive health and quality of life outcomes [10-14].
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ACCEPTED MANUSCRIPT As the absence of ASL from hepatocytes causes ASA-related hyperammonemia [12, 15, 16], liver-targeted gene therapy has the potential to benefit patients with severe disease. Adenoassociated virus (AAV)-mediated gene therapy has demonstrated both therapeutic efficacy and low immunogenicity in murine models for the treatment of other urea cycle disorders (e.g.,
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ornithine transcarbamylase deficiency, arginase deficiency, and citrullinemia) [17-23].
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However, ASA presents a unique challenge as the enzyme is essential not only for the removal
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of waste nitrogen via the urea cycle, but also for the clearance of argininosuccinic acid through its conversion to arginine. The buildup of argininosuccinic acid is thought to cause some of the
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unique symptoms of the disease, including hepatic and cognitive deficits [4, 5]. Based on results
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from patients with ASA who have received liver transplants [15, 16, 24], liver-targeted gene therapy is uniquely positioned to restore the urea cycle and increase quality of life without the
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need for a highly invasive procedure or a continued drug regimen. In this study, we developed a
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gene therapy for ASA using an AAV8 vector expressing the human ASL gene under the control of the liver-specific thyroxine-binding globulin (TBG) promoter, and examined therapeutic
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efficacy in both neonatal and adolescent ASA hypomorphic mice.
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ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1. Mice ASA hypomorphic mice on a C57BL/6 background were acquired from The Jackson Laboratory (Bar Harbor, ME) and a colony was maintained at the University of Pennsylvania under specific All animal procedures and protocols were approved by the
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pathogen-free conditions.
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Institutional Animal Care and Use Committee of the University of Pennsylvania.
2.2. Vectors
promoter
with
a
(AAV8.TBG.hASLco.bGH).
bovine
growth
hormone
(bGH)
polyA
signal
sequence
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TBG
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AAV8 vectors expressing human codon-optimized ASL were designed under the control of a
AAV vectors were produced by the Penn Vector Core at the
2.3. Neonatal gene therapy
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University of Pennsylvania, as previously described [25].
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During the first 24 h following birth, ASA hypomorphic pups (n=4 low dose, n=7 high dose)
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received intravenous (IV) administration of 1010 or 1011 genome copies (GC)/mouse of AAV8.TBG.hASLco.bGH via the temporal facial vein.
Mice were weighed throughout the
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study and genotyped upon weaning. Mice were sacrificed upon reaching median survival, and tissues were collected for histology.
2.4. Adolescent gene therapy ASA hypomorphic mice four to five weeks of age received IV administration of 1013 or 6x1013 GC/kg of AAV8.TBG.hASLco.bGH via the retro orbital vein (n=8).
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Mice were weighed
ACCEPTED MANUSCRIPT throughout the study.
Sub-mandibular bleeds were performed weekly to collect plasma for
monitoring liver transaminases and urea cycle metabolites.
2.5. Serum analyses
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Plasma samples were submitted to Antech Diagnostics (Irvine, CA) for analysis of liver
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transaminases and Charles River Laboratories (Worcester, MA) for analysis of arginine,
acetonitrile.
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citrulline, and argininosuccinic acid by LC-MS/MS following protein precipitation with Mice were sacrificed after three months and tissues were collected for
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biodistribution and histology.
2.6. ASL immunohistochemistry
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Tissues were fixed in formalin for a minimum of 24 h and then paraffin embedded. Sections
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were deparaffinized through an ethanol and xylene series, boiled for 6 min in 10 mM citrate buffer (pH 6.0) for antigen retrieval, and sequentially treated with 2% H 2O2 (15 min),
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avidin/biotin blocking reagents (15 min each; Vector Laboratories, Burlingame, CA), and Sections were then
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blocking buffer (10 min; 1% donkey serum in PBS + 0.2% Triton).
incubated with a rabbit serum against ASL (1 h; HPA016646, Sigma-Aldrich, St. Louis, MO)
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and biotinylated secondary anti-rabbit antibodies (45 min; Jackson ImmunoResearch, Westgrove, PA) diluted in blocking buffer at the manufacturer’s recommended concentration. A Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA) was used according to the manufacturer's instructions with 3,3'-diaminobenzidine as the substrate to stain bound antibodies.
2.7. Sirius Red and HE
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ACCEPTED MANUSCRIPT To detect liver fibrosis, Sirius red staining was performed on paraffin sections. Deparaffinized sections were stained for 90 min in a solution containing 0.1% (w/v) Direct Red (Sigma) and 4% (w/v) picric acid (Sigma-Aldrich, St. Louis, MO), and then washed with 0.01 N HCl (2 x 1 min), dehydrated through an ethanol and xylene series, and coverslipped. Paraffin sections were
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stained with hematoxylin and eosin (H&E) according to standard protocols.
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2.8. Biodistribution
Liver samples were frozen on dry ice at the time of necropsy, and DNA was extracted using the
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QIAamp DNA Mini Kit (Qiagen, Valencia, CA). Detection and quantification of vector GCs in
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extracted DNA were performed by real-time polymerase chain reaction, as previously described [26]. Briefly, genomic DNA was isolated, and vector GCs were quantified using primers/probes
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designed against the promoter sequence of the vector. Quantification of GCs from liver was
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2.9. ASL activity assay
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performed on one liver sample from each mouse.
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Liver (25-30 mg) was added to 200 µl of cold homogenizing buffer containing 50 mM phosphate buffer (pH 7.5) and proteinase inhibitors (EDTA-free proteinase inhibitor cocktail; Roche, Basel,
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Switzerland), and homogenized using Tissue Lyser II (Qiagen, Valencia, CA), at a frequency of 30 for 30 seconds.
Homogenates were centrifuged at 10,000 x g for 20 min at 4ºC, and
supernatants were kept frozen at 80ºC. Lysate (2 µl) was added to 48 µl of 50 mM phosphate buffer (pH 7.3), 3.6 mM argininosuccinic acid (Sigma Aldrich, St. Louis, MO). The reaction was incubated at 37ºC for 1 h and stopped by heating at 80ºC for 20 min. Fumarate was measured with Fumarate Assay Kit (Sigma Aldrich, St. Louis, MO) per the manufacturer’s
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ACCEPTED MANUSCRIPT specifications using 5 µl of reaction sample mixture.
2.10. Statistical analysis Comparison of survival was performed by logrank test with each experimental group compared
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to uninjected control groups, unless otherwise stated. Other parameters were compared by one-
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way analysis of variance (ANOVA) with Dunnett’s multiple comparison test comparing each
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group to the untreated wild-type control group, unless otherwise stated. *p < 0.05, **p < 0.01,
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***p < 0.001.
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3. Results
3.1. An ASA hypomorphic mouse model enables the study of both neonatal and adolescent mice
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To evaluate our gene therapy approach, we first considered which of the two ASA mouse models
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to use for our studies. A knockout model (Asl -/-) was created by replacing exons 8 and 9 with a 1,400 bp neomycin cassette, resulting in a frame shift in the mRNA beginning with exon 10 [27].
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All Asl-/- mice have elevated plasma ammonia, argininosuccinic acid, and citrulline, as well as
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low plasma arginine. However, these mice die within 48 hours of birth, so this model can only be used to study treatment of a neonatal cohort. As an alternative, an ASA hypomorphic mouse
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model has been developed, where a 1,200 bp neomycin cassette was inserted into intron 9, resulting in reduced, but not ablated, mRNA levels and slightly prolonged survival [6]. These mice have the same characteristic variations in amino acids and liver metabolites, and they display a sparse fur coat. Therefore, for the current study we used the ASA hypomorphic mouse. We confirmed that these mice have a mean survival of 22 days (Figure 1A). Homozygous ASA hypomorphic pups were indistinguishable from heterozygous or wild-type littermates until five
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ACCEPTED MANUSCRIPT days after birth, at which time the hypomorphic mice failed to develop a fur coat and were visibly smaller.
3.2. Prophylactic AAV8 gene therapy extends survival of neonatal ASA hypomorphic mice
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We next evaluated AAV8 therapy in neonatal mice. We performed IV injections via the temporal
We genotyped mice after weaning and monitored the vector-administered
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24 hours of birth.
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facial vein with 1010 or 1011 GC/mouse of the AAV8.TBG.hASLco.bGH vector within the first
ASA hypomorphic mice for survival. Vector administration at both doses significantly increased
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survival compared to untreated ASA hypomorphic mice (p < 0.01; Figure 1B). The increase in
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median survival was dose dependent, with a median survival of 165 days for the 1011 GC/mouse group compared to 136 days for the 1010 GC/mouse group. Weight gain for vector-administered
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ASA hypomorphic mice was comparable to wild-type littermates throughout the study, with the
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exception of the female vector-administered hypomorphic mice at the last time point evaluated
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(day 63; Figures 1C).
We used
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Once the mice reached median survival, we euthanized and necropsied them.
immunohistochemistry (IHC) of the liver to visualize ASL protein expression (Figure 2). IHC
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revealed a small number of strongly stained positive cells. The low level of transgene expression observed in these adolescent mice was expected, as the rapid proliferation of hepatocytes during early development has been shown to significantly dilute non-integrating vector genomes [2830].
Despite the low level of transgene expression observed at the time of necropsy, ASL
activity was sufficient to allow all vector-treated ASA hypomorphic mice to live beyond the 22 day mean survival of untreated ASA hypomorphic mice. However, as expected without re-
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ACCEPTED MANUSCRIPT administration, we confirmed that vector dilution as the animal grew led to reduced efficacy and resulted in premature death (Figure 1B).
3.3. AAV8 gene therapy treatment extends survival, increases weight, and normalizes serum
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transaminase levels in adolescent ASA hypomorphic mice
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In addition to a prophylactic gene therapy model following neonatal administration, we
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investigated the potential treatment effects of AAV8 gene therapy in adolescent ASA hypomorphic mice. We administered AAV8 to 30-day-old adolescent mice via the retro orbital
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vein; the standard adolescent mouse IV administration route via the tail vein could not be used
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for these adolescent ASA hypomorphic mice as their average weight was 8.8 g. We collected plasma from the first cohort of female mice for baseline metabolite and transaminase evaluation
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prior to IV administration with 6x1013 GC/kg of vector.
Mice that had been bled had high
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mortality rates prior to or shortly after vector administration, likely due to reduced blood volume and were omitted from the survival data set for this cohort (Figure 3A). Therefore, we did not
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determine baseline values for the other cohorts. Further cohorts of adolescent ASA hypomorphic
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mice were either untreated or administered IV with 1013 GC/kg of vector. Survival of mice in all vector-administered cohorts increased compared to untreated ASA hypomorphic mice (p <
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0.001; Figure 3A). Mean survival was extended to 91 days in the low-dose female group, with the study terminated before the high-dose female and low-dose male cohorts had lost enough mice to determine mean survival (Figure 3A). We observed a sex difference in survival, with untreated female ASA hypomorphic mice having increased mean survival compared to untreated male ASA hypomorphic mice (p < 0.001). However, following low-dose vector administration, male mice showed increased survival and weight gain compared to female mice, which could
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ACCEPTED MANUSCRIPT possibly be due to an androgen-dependent, species-specific effect on AAV transduction of the liver as previously described [31]. We observed a dose-dependent increase in weight gain; however, even mice administered 6x1013 GC/kg of vector did not match wild-type body weights (Figure 3B). All vector-treated groups had reductions in plasma alanine aminotransferase (ALT)
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and aspartate aminotransferase (AST) compared to untreated controls (Figure 3C and 3D).
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3.4. AAV gene therapy normalizes plasma argininosuccinic acid and citrulline levels in ASA
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hypomorphic mice
AAV gene therapy has the unique potential to restore the urea cycle in hepatocytes without the Therefore, we evaluated the effect of our AAV8 vector on
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need for a liver transplant.
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metabolites associated with ASA and other aspects of the urea cycle. Argininosuccinic acid is broken down into arginine and fumarate by the ASL enzyme, leading to elevations in
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argininosuccinic acid and citrulline (a substrate of the reaction upstream of ASL), and reductions
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in arginine.
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Plasma citrulline was elevated to 474 ± 155 µmol in the hypomorphic mice compared to 58.3 ± In the high-dose female and low-dose male
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14.4 µmol in the wild-type mice (Figure 4A).
groups, we saw a dramatic reduction of citrulline in plasma, resulting in no significant difference
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in citrulline levels compared to wild-type littermates indicating correction of the metabolite (Figure 4A). We did not observe an effect of the low dose of vector on plasma citrulline levels in the female mice. Plasma argininosuccinic acid, which is the metabolite that differentiates ASA from other urea cycle disorders and is thought to play a role in the unique symptoms of the disease, was elevated from 163.6 ± 60.0 µmol in the hypomorphic mice compared to 1.53 ± 1.13 µmol in the wild-type mice (Figure 4B). Plasma argininosuccinic acid was corrected to normal
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ACCEPTED MANUSCRIPT levels in the high-dose female group (Figure 4B), but remained elevated in both the male and female low-dose cohorts, indicating a steep dose effect.
Wild-type mice had plasma arginine levels in the range of 45-185 µM with a mean of 95 µM Characterization of arginine levels in the ASA hypomorphic mouse was
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(Figure 4C).
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complicated because of less dramatic differences between values in normal and ASA
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hypomorphic mice, and the early mortality of the mice in the absence of treatment. In fact, at the initiation of the study ASA hypomorphic mice had arginine levels similar to the wild type (75
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µM) (Figure 4C). However, at day 35 we observed significant reductions in arginine levels from
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wild-type mice compared to male and female hypomorphic cohorts treated with 1013 GC/kg of AAV8 vector, suggesting incomplete correction. However, the extent of the therapeutic affect is
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unclear as we do not have samples from untreated hypomorphic mice at this age group to analyze
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(Figure 4C). Incomplete correction may also be due to the liver-restricted nature of our therapy,
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synthesis in the body.
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where to correct arginine we would want to target to the kidney, the primary site of arginine
Upon termination of the study, we sacrificed the mice and harvested liver for determination of
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ASL protein distribution, liver disease progression, and vector GCs. Female ASA hypomorphic mice administered with the high dose of vector had a strong presence of ASL protein in the liver as measured by IHC (Figure 5A). Male and female mice dosed with the low dose showed similar levels of staining.
Activity of ASL protein in liver lysate of high-dose females as
measured by an activity assay was on average 25% of wild-type levels and statistically significantly higher than untreated hypomorphic mice, which had a mean activity of 3% (Figure
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ACCEPTED MANUSCRIPT 5B). The activity in mice administered with the low vector dose was not statistically higher than in the untreated hypomorphic mice.
Vector genome copy analysis on liver samples demonstrated that ~four-fold higher vector GCs
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were present in hepatocytes in the high-dose cohort compared to the low-dose groups (Figure
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5C). These results demonstrate that with sufficient transduction of liver by an ASL gene therapy
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vector, survival in the ASA hypomorphic mouse model can be greatly extended.
This is
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correction of metabolite markers to normal levels.
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concurrent with a reduction in liver transaminases indicating reduced hepatic toxicity and the
The primary histopathologic findings in the liver of ASA hypomorph mice that received either
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low- or high-dose vector included variable hepatocellular changes consisting of enlargement of
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both the nucleus and cytoplasm (karyocytomegaly) with and without individual hepatocellular necrosis and regeneration (indicated by increased incidence of mitotic figures). The more severe
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cases exhibited hepatocellular dissociation with and without lobular collapse. Other findings
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included mixed cellular infiltrates, fibrosis, and higher scores of bile duct and oval cell hyperplasia indicating a more severe phenotype (Figure 6). H&E staining revealed that low-dose
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male mice (Figure 7A and 8A) appeared to have more severe histopathological events than the equivalently dosed female cohort (Figure 7B); by contrast, the high-dose female group trended better than their low-dose counterpart (Figure 7C and 7D).
We observed minimal
histopathologic findings in untreated ASA hypomorphic mice compared to dosed cohorts (Figure 7E). We believe this result is due to the early death of these untreated mice (~ study day 15), as all of the histology of dosed groups were generated from samples taken at the termination of the
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ACCEPTED MANUSCRIPT study (study day 98). In addition, we independently scored the liver fibrosis based on location and amount of hepatic infiltration on sections using Sirius red. Although we observed a range of scores for both doses, animals from the low-dose male cohort group (Figure 8B) more commonly showed the presence of and higher scores for fibrosis, compared to the low-dose (Figure 8C) or Again, untreated mice did not develop observable
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histopathology due to the limited time for it to develop (Figure 8E).
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high-dose (Figure 8D) female cohorts.
4. Discussion
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ASA, caused by a deficiency in ASL activity, is characterized by the dysregulation of the urea
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cycle that inhibits arginine synthesis and nitric oxide production [4]. Here, we showed that delivery of the ASL gene into hepatocytes increased survival and weight gain of ASA
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hypomorphic mice. Median survival for ASA hypomorphic mice was 22 days post birth due to a
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failure to thrive marked by an increase in liver transaminases and elevations in urea cycle metabolites. We were able to increase survival of these mice in a dose-dependent manner via
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neonatal administration of vector through the facial vein. However, treated mice did not gain
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weight to an equivalent degree as their wild-type littermates; liver histology suggested that dilution of vector genomes occurred with growth of the animal, which obscures regional
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transduction differences, consistent with what has been previously reported [28-30]. We believe that this vector dilution contributed to the early death of treated hypomorphic mice, where ASL expression necessary for survival may have dropped lower than a critical threshold resulting in a buildup of toxic metabolites. This will be a critically important phenomenon in advancing gene therapy for infant patients with ASA, as readministration of gene therapy may be necessary. Successful readministration of vector to the liver can prove difficult due to circulating
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ACCEPTED MANUSCRIPT neutralizing antibodies to the vector capsid that would develop after the initial administration. However, recent advances in immune suppression have shown potential for either preventing AAV antibody development or reducing the circulating antibodies to other AAV serotypes,
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allowing for repeat dosing [32, 33].
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To determine the efficacy of ASA gene therapy in more developed livers, we administered
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vector via a retro orbital IV injection to adolescent mice. These mice exhibited both increased survival and weight gain, although only the high dose resulted in survival similar to wild-type
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littermates. One potential cause of the continued early morbidity in treated adolescent mice may
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be the vector genome dilution, because although these mice are older than the newborn treatment groups, they have not yet reached full size. Indeed, based on our monitoring of weight of
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untreated ASA hypomorphic mice, a successful start to the therapy may ultimately result in an
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attenuation of its efficacy as it allows the treated mice to increase in weight rapidly.
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Cirrhosis of the liver is one of the hallmarks of ASA, affecting individuals more commonly than
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other urea cycle disorders, and is a primary reason that patients undergo liver transplant [3]. We did not observe liver pathology in untreated ASA hypomorphic mice following review of liver
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sections by a board-certified veterinary pathologist potentially because the affected mice died before cirrhosis could develop. However, we did observe extensive histopathology in some treated groups, especially the low-dose male cohort.
The low-dose female cohort had a
comparatively less severe phenotype that was further reduced in the high-dose cohort. These results could be indicative of a sex bias in treatment efficacy toward more correction in female mice; however, based on metabolite analysis of citrulline we might predict that the opposite is
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ACCEPTED MANUSCRIPT occurring. Another possibility is that ASA liver pathology is more severe in male mice. This line of inquiry requires further understanding of the natural history of the ASA hypomorph, as no reports in the literature suggest a sex difference in the severity of ASA liver cirrhosis. Overall, the severity of histopathological findings in the female cohorts did seem to improve in a dose-
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dependent manner indicating that gene therapy could be effective in treating one of the primary
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reasons patients with ASA require liver transplant [3]. We also observed reduced transaminase
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levels in all treated mouse groups at most time points studied. Furthermore, mice in the highdose group showed a trend for corrected arginine levels, indicating that AAV gene therapy might
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correct arginine deficiency, which is not corrected sufficiently with liver transplant [3, 4]. The
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high-dose group also achieved normalization of plasma argininosuccinic acid. This encouraging
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result suggests that argininosuccinic acid is being cleared from the liver.
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Citrulline, a metabolite upstream of ASL, was normalized in both high-dose administered females and low-dose administered males.
Evidence suggests that an androgen-dependent
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mechanism enhances transduction in male mice (27). However, we did not observe a difference
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in the vector genome copy number between low-dose groups based on sex, suggesting a potential
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difference in disease severity based on sex [31].
One of the major difficulties facing gene therapy treatment of ASA is the importance of ASL in non-hepatic metabolic pathways outside of the urea cycle and how they may negatively impact therapeutic efficacy [13, 14]. ASL is required for both cleavage of argininosuccinic acid into arginine in the kidneys, providing a major of source of serum arginine [34], and production of nitric oxide by endothelial cells lining the arteries [35]. Helper-dependent adenoviral vector
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ACCEPTED MANUSCRIPT gene therapy for this hypomorphic mouse model of ASA showed similar improvements in repair of the urea cycle compared to our vector, but was unable to overcome other disease phenotypes associated with non-liver expression of ASL; we believe this would not be different for our gene therapy approach [36]. Initial longitudinal studies of orthotopic liver transplants for patients with
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ASA, whose outcome a liver-targeted gene therapy could mimic, displayed a less positive
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outcome compared to other urea cycle disorders [14]. A debate has existed among researchers as
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to whether this low efficacy is due to insufficient arginine, loss of nitric oxide synthesis contributing to the cirrhosis of the liver, or buildup of argininosuccinic acid [12, 37]. However,
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more recent studies of liver transplants have shown stronger positive outcomes [12, 15, 16].
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Taken together, it would seem prudent to consider continuing arginine supplementation after gene therapy, and with early treatment and improved care outcomes it may come to resemble
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more recent transplant success.
5. Conclusions
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In summary, we have demonstrated the feasibility of gene therapy for ASA using AAV8 vector
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technology. However, both routes of administration (IV via the temporal facial vein in newborns and retro orbitally in adolescent mice) pose some challenges. Neonatal administration was able
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to extend survival compared to untreated ASA hypomorphic mice, but not to the full lifespan of wild-type mice. This could be solved with readministration of the gene therapy vector at a later time point, an important consideration for future development.
Adolescent vector
administration, while able to correct metabolites at the highest dose tested, was less efficacious at a lower dose.
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ACCEPTED MANUSCRIPT Acknowledgments We thank Deirdre McMenamin, Christine Draper, Hongwei Yu, and Meardey So for invaluable technical assistance.
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Disclosures
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J.M. Wilson is an advisor to, holds equity in, and has a sponsored research agreement with
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REGENXBIO and Scout Bio; he also has a sponsored research agreement with Ultragenyx, Biogen, and Janssen, which are licensees of Penn technology. In addition, he has sponsored
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research agreements with Precision Biosciences and Moderna Therapeutics.
J.M.W. is an
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inventor on patents that have been licensed to various biopharmaceutical companies.
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Funding Source
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This study was funded by internal funding from the University of Pennsylvania.
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ACCEPTED MANUSCRIPT Figure Legends
Figure 1. AAV8 gene therapy extends survival of an ASA hypomorphic mouse model. (A) A survival curve of ASA hypomorphic mice is shown (n = 81). (B) ASA hypomorphic mice
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were injected IV via the temporal vein with 10 10 GC/mouse (n=5) or 1011 GC/mouse (n=6) of
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AAV8.TBG.hASLco and monitored for survival. Comparisons to uninjected ASA hypomorphic
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mice (solid line) are shown. The weight of female (C) and male (D) ASA hypomorphic mice administered with 1011 GC/mouse of AAV8.TBG.hASLco was monitored, and compared to
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wild-type (WT) mice and ASA hypomorphic mice that did not receive gene therapy. Error bars
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= standard error of the mean (SEM); **p < 0.01, ***p <0.001.
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Figure 2. ASL protein expression in the liver of newborn injected mice.
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ASA hypomorphic mice were injected IV via the temporal vein with 10 10 GC/mouse (n=5) or 1011 GC/mouse (n=6) of AAV8.TBG.hASLco.
Mice were necropsied after cohort-specific
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median survival was reached. Livers were harvested and IHC was performed for detection of the
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ASL protein, and compared to wild-type (WT), heterozygous (Het), and untreated ASA hypomorphic mice.
Central veins are denoted by asterisks.
Representative sections from
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different mice in each cohort are shown.
Figure 3.
Sex difference in survival and efficacy of AAV8 gene therapy in ASA
hypomorphic mice. ASA hypomorphic mice were injected IV via the retro orbital vein on P30 with 1013 or 6x1013 GC/kg of AAV8.TBG.hASLco (n=8/group). Mice were monitored for survival (A) and weight
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ACCEPTED MANUSCRIPT (B). Comparisons to uninjected ASA hypomorphic mice are shown (female, n=3; male, n=4). Mice were bled and plasma was analyzed for the liver transaminases AST (C) and ALT (D). Comparison of values to uninjected ASA hypomorphic mice (no vector) at the same time point
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are shown (n=5). Error bars = SEM; **p < 0.01, ***p <0.001.
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Figure 4. High-dose AAV8 gene therapy corrects liver metabolism in ASA hypomorphic
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mice.
ASA hypomorphic mice were injected IV via the orbital vein on P30 with 1013 or 6x1013 GC/kg
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of AAV8.TBG.hASLco (n=8/group). Mice were bled and plasma was analyzed for components
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of the urea cycle, including (A) citrulline, (B) ASA, and (C) arginine. Comparisons to levels in wild-type (WT) mice are shown (n=22), ASA hypomorph control (n=12). Error bars = SEM; ns,
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not significant; *p < 0.05, **p < 0.01, ***p <0.001.
Figure 5. Detection of ASL protein expression and genome copies in the liver of adolescent
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injected mice.
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ASA hypomorphic mice were injected IV via the retro orbital vein at P30 with 1013 (n=8/sex) or 6x1013 GC/kg of AAV8.TBG.hASLco (n=8/group). (A) Livers were harvested and IHC was
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performed for detection of the ASL protein. Each section is a representative sample from a different mouse within each cohort.
Central veins are denoted by asterisks.
(B) Liver
homogenates were assayed for ASL activity compared to wild-type levels (data presented as percentage of wild-type activity). Analysis of activity was performed by a one-way ANOVA with Dunnett’s multiple comparison test comparing each group to uninjected ASA hypomorphic mice (no vector). (C) Following extraction of DNA from liver, vector GCs were determined.
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ACCEPTED MANUSCRIPT Error bars = SEM; ***p < 0.001.
Figure 6.
Histopathologic findings in the liver of a mouse model of ASL administered
either a low (1x1013 GC) or high dose (6x1013 GC) of AAV.
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ASA hypomorphic mice were injected IV via the retro orbital vein at P30 with 10 13 (n=8/sex) or
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6x1013 GC/kg of AAV8.TBG.hASLco (n=8/group). Livers were harvested and H&E and Sirius
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red staining were performed for observation of histopathology. Findings from each category were ranked from 0-5 for hepatocellular changes with 0 being no finding, 5 being marked
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karyocytomegaly (>90% of hepatocytes within a lobule) with lobular collapse and numerous
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single-cell necrosis, and 1-3 denoting increasing severity.
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Figure 7. Histopathologic findings in the liver of a mouse model of ASL administered with
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either a low (1x1013 GC) or high dose (6x1013 GC) of AAV. The images depict representative trends of the histopathological findings for each dose group.
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(A) A male mouse received a low dose of vector and exhibited grade 4 hepatocellular changes
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characterized by severe karyocytomegaly (51-90% of hepatocytes within a lobule) with extensive hepatocellular dissociation and frequent individual cell necrosis; however, generally
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normal hepatocellular architecture was maintained. This animal also had oval cell (grade 3) and bile duct (grade 1) hyperplasia.
(B) A female mouse also received a low dose of vector;
however, hepatocellular changes were less severe (grade 2) characterized by mild karyocytomegaly (< 10% of hepatocytes within a lobule) with normal hepatic architecture and no individual cell necrosis.
This animal had no oval or bile duct hyperplasia.
(C) The
histopathologic findings were similar in a female mouse administered with a high dose of vector.
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ACCEPTED MANUSCRIPT (D) However, other animals in this dose group had no significant liver findings, as depicted in the displayed mouse. H&E, Scale bar = 100 µm.
Figure 8.
Variable fibrosis was observed in the liver of a mouse model of ASL
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administered with either a low (1x1013 GC) or high dose (6x1013 GC) of AAV.
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Sirius red- and H&E-stained liver sections were scored based on location and amount of hepatic
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infiltration. The severe cases (grade 3) were mostly in the low-dose group and were clearly visible on H&E staining. (A) In a male mouse from the low-dose cohort, the liver had regions of
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bridging fibrosis between portal and occasionally centrilobular areas with architectural disruption
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and individualization of hepatocytes. The presence of hepatocellular mitotic figures, indicative of regeneration, was frequently observed in these areas (inset, scale bar = 20 pixel). In these
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cases, the fibrosis was frequently accompanied by oval cell and bile duct hyperplasia. Sirius red
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staining highlighted the fibrosis in less severely affected sections. (B) A male mouse in the lowdose group exhibited regional bridging portal fibrosis (grade 2) with no architectural distortion,
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(C) whereas only periportal fibrosis (grade 1) was observed in a female mouse from the high-
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dose group. (D) No fibrosis was observed in another female mouse from the high-dose group.
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Sirius red, Scale bar = 100 µm.
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