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Division of Gene Therapy and Regulation of Gene Expression, Cima Universidad de Navarra, Pamplona, Spain (LO, Technician; DS, Technician; GGA, Principal Investigator)
Division of Gene Therapy and Regulation of Gene Expression, Cima Universidad de Navarra, Pamplona, Spain (LO, Technician; DS, Technician; GGA, Principal Investigator)Current affiliation: Translational Research, Viralgen V.C., San Sebastián, Spain (DS, Scientist)
Liver Unit, Internal Medicine Department. Clínica Universidad de Navarra and Hepatology Program, CIMA, Universidad de Navarra, Pamplona, Spain (JA, Principal Investigator)Division of Medicine - Gastroenterology and Hepatology Department, University of Pittsburgh. Pittsburgh, PA USACentro de Investigacion Biomedica en Red (CIBER-Ehd), Madrid, Spain
Gene therapy redosing may be necessary to maintain efficacy in pediatric patients with inborn hepatobiliary disorders
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Coadministration with tolerogenic nanoparticles carrying rapamycin enabled an efficacious second administration of AAV
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Only following a second AAV dose after treatment with AAV plus ImmTOR did infant PFIC3 mice exhibit a therapeutic effect
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Repeat administration also prevented the appearance of liver malignancies in mice prone to hepatocellular carcinoma
Abstract
Background and Aims
Gene therapy using recombinant adeno-associated virus (rAAV) vector carrying MDR3 coding sequence (AAV8-MDR3) represents a potential curative treatment for progressive familial intrahepatic cholestasis type 3 (PFIC3), which presents in early childhood. However, patients with the severest form of PIFC3 should receive treatment early after detection to prevent irreversible hepatic fibrosis leading ultimately to liver transplantation or death. This represents a challenge for rAAV-based gene therapy since therapeutic efficacy is expected to wane as rAAV genomes are lost due to hepatocyte division, and the formation of AAV-specific neutralizing antibodies (NAbs) precludes readministration. Here, we tested a strategy of vector readministration in infant PFIC3 mice with careful evaluation of its oncogenicity—a particular concern surrounding rAAV treatment.
Methods
AAV8-MDR3 was readministered to infant Abcb4−/− mice two weeks after a first dose coadministered with tolerogenic nanoparticles carrying rapamycin (ImmTOR) given at two weeks of age. Eight months later, long-term therapeutic efficacy and safety were assessed with special attention paid to the potential oncogenicity of rAAV treatment.
Results
Coadministration with ImmTOR mitigated the formation of rAAV-specific NAbs and enabled an efficacious second administration of AAV8-MDR3, resulting in stable correction of the disease phenotype, including a restoration of bile phospholipid content and healthy liver function; as well as the prevention of liver fibrosis, hepatosplenomegaly, and gallstones. Furthermore, efficacious repeat rAAV administration prevented the appearance of liver malignancies in an animal model highly prone to develop hepatocellular carcinoma.
Conclusions
These outcomes provide strong evidence for rAAV redosing through coadministration with ImmTOR, as it resulted in a long-term therapeutic effect in a pediatric liver metabolic disorder, including the prevention of oncogenesis.
Lay Summary
Redosing of gene therapy for inborn hepatobiliary disorders may be essential as effect wanes during hepatocyte division and renewal, particularly in pediatric patients, but the approach may carry long-term risks of liver cancer. Viral vectors carrying a therapeutic gene exerted a durable cure of progressive familial intrahepatic cholestasis type 3 in infant mice and reduced the risk of liver cancer only following a second administration.
NDW, IRG, JPC and GGA are employees and stockholders of Vivet Therapeutics. TKK is an employee of Selecta Biosciences. All other authors declare no conflict of interest.
Financial support statement:
This study was funded by Vivet Therapeutics and Selecta Biosciences and via a Government of Navarra PhD grant ‘Doctorados industriales 2019–2021’.
Data Availability Statement:
The data generated from the studies are available from the corresponding author on reasonable request.
Author contributions:
Study conception and design: NDW, JPC, TKK and GGA; Acquisition of data: NDW, LO, IRG and DS; Analysis and interpretation of data: NDW, LO, GGP, JA and GGA; Statistical analysis: NDW; Drafting of the manuscript: NDW, IRG, GGP, TKK and GGA; Important intellectual contributions during the study as well as critical revision of the manuscript: JPC, TKK, JA and GGA; Study supervision: NDW and GGA.
Introduction
Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare metabolic disorder that arises from genetic alterations in the ABCB4 gene
, which encodes multidrug resistance protein 3 (MDR3), a floppase involved in the translocation of phosphatidylcholine (PC) from hepatocytes into the bile
. Without sufficient PC transport into bile, the concentration of free bile acids is highly elevated, increasing the detergent properties of the bile and causing toxic injury to the canalicular membrane of hepatocytes and cholangiocytes, resulting in hepatic parenchyma and bile duct damage. Biliary obstruction may result from long-term damage of biliary epithelium and from formation of gallstones due to reduced phospholipid concentration in the bile. This pathology leads to cholestasis, cirrhosis and ultimately end-stage liver disease, which is fatal in the absence of liver transplantation
. This vector is comprised of a codon-optimized version of the ABCB4 gene encoding human MDR3 isoform A protein under control of the liver-specific alpha-1 antitrypsin promoter flanked by AAV2 inverted terminal repeats housed in an AAV serotype 8 capsid. The therapeutic efficacy of AAV8-MDR3 was tested in an FVB background strain mouse model for PFIC3 that lacks the first two exons of the Abcb4 gene resulting in a total lack of MDR2 protein (the mouse ortholog of human MDR3) expression and activity
. This model, in the absence of treatment, reliably presents many of the same phenotypic manifestations of MDR3 deficiency observed in children, such as elevated serum liver enzymes, hepatosplenomegaly, reduced biliary phosphatidylcholine, cholelithiasis
. FVB.Abcb4–/– mice treated with AAV8-MDR3 showed a complete reversal of this pathology when administered at two or five weeks of age, normalizing levels of disease biomarkers to those observed in wild-type (wt) controls. However, a non-inconsequential shortcoming of the therapy was the loss over time of the therapeutic effect. This appeared to be driven by the loss of MDR3 expression resulting in an increase in liver damage and hepatocyte regeneration and the creation of a positive feedback loop. The appearance of PFIC3 disease manifestations in a subset of the study animals was accompanied by hepatosplenomegaly, a complete reversion of bile PC to untreated levels and the appearance of liver fibrosis. Thus, evidence from these first studies supported a common assumption in gene therapy approaches for pediatric and degenerative liver diseases that readministration may be essential to maintain the phenotypic correction of the disorder
Despite its potential in achieving long-term therapeutic benefit, readministration of an AAV-based gene therapy vector would have to circumvent AAV-specific neutralizing antibodies (NAbs)
Effects of transient immunosuppression on adenoassociated, virus-mediated, liver-directed gene transfer in rhesus macaques and implications for human gene therapy.
produced as a consequence of the first administration. One approach is the use of vectors with different capsid serotypes for subsequent administrations
, which has the drawbacks of potentially reduced transduction efficiency and the required development of multiple vectors. The use of techniques to remove antibodies from circulation such as plasmapheresis, immunoadsorption or immunoglobulin G proteases has shown promising results but only for low antibody titers
. Alternatively, an approach that prevents the immune response during the initial vector administration could allow for subsequent rounds of the same AAV treatment. Paramount to this approach is the prevention of AAV-specific NAbs
Tolerogenic nanoparticles containing rapamycin (ImmTOR) have demonstrated the ability to induce durable immunological antigenic tolerance when co-administered with biologic drugs, including enzymes, monoclonal antibodies, and AAV
. ImmTOR nanoparticles, when injected intravenously, are taken up by antigen presenting cells such as dendritic cells or macrophages in the spleen and liver
. Immunological tolerance results from the induction of antigen-specific regulatory T cells, lower activation of B cells, and, therefore, an inhibition of the production of antibodies
. Therefore, it was hypothesized that co-administration of ImmTOR nanoparticles with AAV8-MDR3 might mitigate formation of specific NAbs against the vector and allow for its readministration to rescue infant PFIC3 mice.
Here, we evaluated this hypothesis with AAV8-MDR3 co-administered with ImmTOR. FVB.Abcb4−/− mice that received readministration following a first dose co-administered with ImmTOR were unique in showing a robust and durable therapeutic effect as shown by the correction of all disease markers and evidence of the prevention of NAb formation from the first AAV treatment. Of particular importance, the AAV8-MDR3 plus ImmTOR treatment was administered to mice at 2 weeks of age, demonstrating to our knowledge the first instance of a successful immunotolerogenic effect and vector redosing in study animals that are representative of young pediatric patients. What is more, the treatment rather than inducing oncogenicity showed a clear inhibitory effect in the formation of liver malignancies.
Materials and Methods
A detailed description of the materials and methods used in this study is included in the supplementary information.
Results
Identification of subtherapeutic dose of AAV8-MDR3
We first performed a dose range finding study of AAV8-MDR3 in two-week-old infant FVB.Abcb4–/– mice of both sexes to identify a subtherapeutic dose that would transiently delay the appearance of cholestasis symptoms. It was predicted that a transient treatment-induced normalization of cholestatic serum biomarkers would indicate a delay in the appearance of liver fibrosis, allowing for a second AAV treatment to be tested for the ability to provide long-term correction of the disease without transduction efficiency being impaired in a fibrotic liver. Mice were treated intravenously with AAV8-MDR3 at doses of 2x1013, 4x1013 and 8x1013 vector genomes (vg)/kg and then followed weekly for levels of cholestasis biomarkers in the serum. While the two lower doses were ineffective at normalizing biomarker levels, the 8x1013 vg/kg dose was transiently effective in reducing serum levels of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), bile acids (BA), and bilirubin (BIL) (Figs. 1A-D; S1A-B). After 11 days of AAV8-MDR3 administration any evidence of a therapeutic effect was lost. Upon sacrifice thirty-one days post-injection (p.i.), none of the treated mice showed any reduction in hepatosplenomegaly (Fig. 1E & F), a characteristic feature of diseased animals, nor any improvement in biliary PC levels (Fig. 1G). Animal body weights were unaffected by treatment (p=0.67, 2-way ANOVA; Fig. S2). The 8x1013 vg/kg dose was thus selected for the next studies.
Fig. 1Single administration of subtherapeutic doses of AAV8-MDR3 in infant FVB.Abcb4-/- mice can exert a transient therapeutic effect. FVB.Abcb4-/- mice (n=3M/2-3F for each treated group; n=2M/1F for Saline) were treated i.v. with AAV8-MDR3 at 2, 4 or 8x1013 vg/kg at 2 weeks of age. (A-D) Liver transaminases, alkaline phosphatase and bile acids in serum are indicated at 7 to 28 days post-AAV treatment. Statistics: 2-way ANOVA; color of asterisks indicates significance of corresponding group vs. WT at indicated timepoint; *, p<.05; **, p<.01; ***, p<.001; ***, p<.0001. ALT, alanine transaminase; AST, aspartate transaminase; ALP, alkaline phosphatase; BA, bile acids; U/L, units/liter. (E-G) Liver (E) and spleen (F) weights as percent of body weight and bile phosphatidylcholine levels (G) at sacrifice, day 31 post-AAV treatment, are shown. Age-matched wild-type mice (n=3M/3F) are shown. Statistics: 1-way ANOVA; *, p<.05; ***, p<.001; ***, p<.0001.
Next, we investigated the immunomodulatory properties of ImmTOR when coadministered with the sub-therapeutic dose of 8x1013 vg/kg AAV8-MDR3. Two-week-old male and female mice were treated i.v. with AAV8-MDR3 with or without coadministration of ImmTOR (equivalent of 15 mg/kg rapamycin). Then, a subset of the study animals received a second dose of AAV8-MDR3 (8x1013 vg/kg) alone 2 weeks later (Fig. S3). AAV co-administration with empty nanoparticles (NP[empty]) served as a vehicle control. Mice were followed for 8 months with periodic analyses of serum biomarkers to determine the progression of the disease.
Only FVB.Abcb4–/– mice treated with AAV8-MDR3 co-administered with ImmTOR at the first dose showed a durable therapeutic effect when a second dose of AAV8-MDR3 alone was administered 2 weeks p.i (Figs. 2A-C; S4). Mice treated with AAV8-MDR3 twice without ImmTOR lost any therapeutic effect after 2 weeks post-treatment since the second administration of AAV8-MDR3 had no apparent effect. Interestingly, mice given one treatment of AAV co-administered with ImmTOR showed a partial and transient therapeutic effect up through 4 weeks post-treatment, but without a second AAV treatment, the effect was lost in all animals, and the serum liver enzyme levels of these mice rebounded to levels seen in other untreated and unsuccessfully treated PFIC3 mice (Figs. 2A-C; S4). Body weights of treated mice were statistically similar to wt mice (p=0.92, 2-way ANOVA; Fig. S5).
Fig. 2Readministration of AAV8-MDR3 when admixed with ImmTOR in infant FVB.Abcb4-/- mice induces a durable therapeutic effect. FVB.Abcb4–/– mice were injected i.v. at two weeks of age with AAV8-MDR3 admixed with ImmTOR and then again two weeks later with AAV8-MDR3 alone. (A-C) Liver transaminases and alkaline phosphatase in serum are indicated up to 32 weeks post-AAV treatment. ALT, alanine transaminase; AST, aspartate transaminase; ALP, alkaline phosphatase; U/L, units/liter. Group size: AAV8-MDR3+ImmTOR / AAV8-MDR3, n=3M/2F; AAV8-MDR3/AAV8-MDR3 and AAV8-MDR3+ImmTOR, n=5M/2F; AAV8-MDR3, n=7M/5F; No AAV, 5M/3F; WT, 3M/3F. Statistics: 2-way ANOVA; color of asterisks indicates significance of corresponding group vs. AAV+ImmTOR / AAV at indicated timepoint; *, p<.05; **, p<.01; ***, p<.001; ***, p<.0001. AAV+ImmTOR / AAV vs. WT, not significant at any point.
The mice receiving AAV8-MDR3 twice with ImmTOR on the first treatment maintained a therapeutic effect up through 32-weeks post treatment with serum ALT, AST, ALP, BA, cholesterol (CHOL), and BIL levels fully normalized to wt controls. Upon study termination, these animals showed liver and spleen sizes equivalent to wt, increased bile PC concentration, and a total absence of liver fibrosis, while all untreated and unsuccessfully treated PFIC3 animals showed clear manifestations of cholestasis (Figs. 3; S6–S10). Notably, the livers of the cured PFIC3 mice were indistinguishable from wt liver tissue in terms of histology and degree of fibrosis (hematoxylin and eosin staining and Sirius Red staining), while all other animals showed extensive bridging fibrosis to severe cirrhosis, enlarged bile ducts and cell infiltrates (Figs. 3E, S6). Quantification of the total amount of collagen in the liver tissue of treated mice (Sirius Red staining), levels of which indicate the extent of fibrosis, was on average 14.8 ± 4.6% that of diseased animals (a 6.8-fold reduction), which was the same as wt mice (14.8 ± 5.0%) (p=2E-5, 2-tailed t-test: treatment vs. all unsuccessfully treated mice). Liver fibrosis was also assessed by quantifying expression of the fibrosis markers collagen type 1 alpha 1 (Col1a1), lysyl oxidase 1 (Lox) and tissue inhibitor of metalloproteinase 1 (Timp1) (Fig. S7) showing significantly lower levels in successfully treated mice, which were similar to wt levels for all three markers (p=0.002, p=0.015, p=0.008, respectively; 2-tailed t-test: treatment vs. all unsuccessfully treated mice).
Fig. 3Phenotypic correction in infant FVB.Abcb4-/- mice following readministration of AAV8-MDR3 admixed with ImmTOR. (A-C) Liver (A) and spleen (B) weights as percent of body weight and bile phosphatidylcholine levels (C) at sacrifice, 32 weeks post-first-AAV treatment, are shown. PC, phosphatidylcholine; 1X or 2X, one or two times. (D) Quantification of liver fibrosis as indicated by Sirius Red staining of liver sections of study animals at sacrifice are shown. Statistics: 1-way ANOVA, *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. (E) Representative images of Sirius Red (above) and hematoxylin and eosin (below) stained liver sections from study animals as indicated. Scalebar = 0.5mm. (F) Hepatocellular neoplasms in KO mice according to percentage of hepatocytes with MDR3 protein expression via IHC or WT mice. Statistics: chi-square test.
Mean bile PC concentration reached 42.6 ± 10.3% wt levels in mice treated with 2× AAV treatment with ImmTOR, while unsuccessfully treated PFIC3 mice had on average only 1.8 ± 3.0% wt levels (p=6E-15) (Fig. 3C). While diseased animals had livers and spleens that were on average 2-fold larger than wt animals (201 ± 37% and 198 ± 53% wt values for liver and spleen sizes, respectively), the mice that received 2× AAV treatment with ImmTOR showed no increase in liver and only slight increase in spleen sizes (104 ± 4% and 119 ± 24% wt values; p=2E-5 and 8E-3 versus all unsuccessfully treated) (Fig. 3A-B). Diseased animals also showed extensively enlarged bile ducts in the liver (Fig. S6) and presented gallstones in the gall bladder and bile duct (39% of unsuccessfully treated PFIC3 mice) (Figs. S8–S9), while none of the mice given 2× AAV treatment with ImmTOR did. Bile duct proliferation was analyzed by cytokeratin 19 (CK19) immunohistochemistry (IHC). Unsuccessfully treated PFIC3 mice showed a clear proliferation of bile ducts that was significantly reduced by 2× AAV treatment with ImmTOR (p=.023, 2-tailed t-test: treatment vs. no AAV control) showing levels similar to those observed in healthy wt mice (Fig. S10). Double immunofluorescence analysis with anti-CK19 and E-cadherin or vimentin showed in all cases that CK19 are e-cadherin positive and vimentin negative. Vimentin-positive cells surrounded CK19-positive cells. However, we observed a very different organization of the morphological structures depending on the treatment. While wt mice and 2× AAV with ImmTOR treated mice showed a clear and well organized bile duct structure surrounded by vimentin-positive cells, in untreated and unsuccessfully treated PFIC3 mice we observed very disorganized cellular distributions (Figs. S11–12). Thus, the reactive cholangiocyte phenotype appeared not to be affected by successful treatment and prevention of cholestasis-related sequelae, as CK19-positive cells exhibited an epithelial phenotype and no epithelial-mesenchymal transition regardless of treatment or disease severity. This phenotype of cholangiocytes in the PFIC3 mice was not different from wt mice.
AAV readministration prevents liver malignancies in PFIC3 mice
, shorter study durations did not allow for an analysis of whether AAV8-MDR3 treatment could prevent or induce the appearance of liver tumors. After 8 months of follow-up in the current study, none of the mice receiving AAV8-MDR3 twice with ImmTOR showed any liver pathology indicative of hepatocellular carcinoma (HCC). Conversely, almost all of unsuccessfully treated animals presented significant liver damage as expected, and half developed atypical hepatocellular neoplasms or dysplastic nodules detected via histopathological analysis (Figs. S9A, S13-15). To assess a correlation between MDR3 expression and the appearance of hepatocellular neoplasms, the mice were binned according to percentage of hepatocytes with MDR3 expression as detected by IHC with a threshold of 15% positivity serving as the cutoff. Neoplasms were detected in 51.9% of animals below this threshold but in none of the animals with MDR3-positive hepatocytes above this threshold (Fig. 3F) (χ2(1) = 4.61, p=.0159). Survival rates were similar for all study groups (Fig. S8C, p=0.55, Mantel-Cox log-rank test). We performed an analysis of hepatocyte proliferation in the liver tissue by detecting Ki-67, a nuclear marker for cell proliferation, via IHC and RT-qPCR. Unsuccessfully treated animals had 0.38 ± 0.47% nuclei that stained positive for Ki-67 vs. 0.054 ± 0.049% for 2× AAV treatment with ImmTOR (p=0.19; Fig. S16), a 7-fold reduction. Mki67 mRNA in unsuccessfully treated PFIC3 mice were 1.9E−3 ± 1.1E−3 copies per transcript copy of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (Gapdh) vs. 2.4E−4 ± 1.7E−4 in mice receiving 2× AAV treatment with ImmTOR (p=4.4E−3; Fig. S16G), an 8-fold reduction. Reduced hepatocyte proliferation is evidence of a lower risk of developing HCC. The phosphorylated histone H2ax (γH2ax) was also quantified via IHC. γH2ax is an indicator of DNA double-strand breaks that has been reported to be a good preneoplasia marker of HCC in these mice
. Results showed 0.93 ± 0.60% γH2ax-positive nuclei in unsuccessfully treated mice compared to 0.20 ± 0.15% in mice given 2× AAV treatment with ImmTOR (p=0.026; Fig. S17), while wt mice had 0.18 ± 0.26% (p=0.94). These results all support the conclusion that AAV8-MDR3 redosing via the use of ImmTOR successfully prevented the development of HCC in PFIC3 mice.
AAV transduction and transgene expression
To gain a measure of the effectiveness in transduction and transgene expression of AAV redosing following coadministration with ImmTOR, AAV genome copy numbers and MDR3 transcripts in liver tissue of sacrificed mice were quantified via qPCR and RT-qPCR, respectively (Fig. 4). FVB.Abcb4-/- mice treated with AAV8-MDR3 only once (either with or without ImmTOR) or twice without ImmTOR showed low AAV transduction at 32 weeks post-treatment with on average 0.0015 ± 0.0013 AAV genome copies per genomic copy of the housekeeping gene Gapdh. In contrast, mice treated with AAV8-MDR3 twice with ImmTOR at the first treatment showed 1.8 log higher AAV genome copies (0.091 ± 0.016 copies; p=2E-19). This transduction efficiency, achieved due to a successful repeat administration of the AAV vector, led to a similar 1.8 log higher level of MDR3 mRNA transcripts in 2× AAV treatment with ImmTOR in comparison to the groups receiving one dose of AAV (with our without ImmTOR) or two doses of AAV without ImmTOR. There were on average 0.54 ± 0.21 MDR3 mRNA copies per Gapdh transcript, while the mice treated with other AAV regimens had only 0.0095 ± 0.0203 copies (p=5E-11). Interestingly, when murine Mdr2 mRNA was quantified in livers of wt mice, 0.053 ± 0.013 copies per Gapdh transcript were determined, 10-fold less than the successfully readministered FVB.Abcb4-/- mice (p=9E-4) (data not shown).
Fig. 4AAV transduction and MDR3 expression after readministration of AAV8-MDR3 admixed with ImmTOR in infant FVB.Abcb4-/- mice. AAV vector genome copy numbers (A) and human MDR3 transcript levels (B) in mouse liver were quantified by qPCR and RT-qPCR, respectively, at sacrifice, 32 weeks post-first-AAV treatment. Values are presented relative to genomic copies or mRNA copies of the housekeeping gene glyceraldehyde 3-phophate dehydrogenase. Gapgh, glyceraldehyde 3-phophate dehydrogenase; 1X or 2X, one or two times. (C) Immunohistochemistry staining of MDR3 protein in mouse liver sections at sacrifice. Scalebar = 100μm. (D) Quantification of percent hepatocytes adjacent to positive MDR3 protein stain. Statistics: 1-way ANOVA, ***, p<0.001; ****, p<0.0001.
Essential for strategies of gene supplementation via gene therapy, in addition to successful transgene delivery, is the correct translation and post-translational processing of the therapeutic protein. Detection of successful MDR3 protein translation and transport to the biliary canaliculi membrane was made via IHC staining of liver sections with an antibody specific for human MDR3. All animals readministered with AAV8-MDR3 following AAV8-MDR3+ImmTOR showed diffuse MDR3 staining throughout the whole liver localized to the biliary canaliculi of hepatocytes (Fig. 4C), while unsuccessfully treated animals showed little to no MDR3 protein. Quantification of the amount of staining revealed a 0.9 log increase due to readministration following AAV8-MDR3+ImmTOR (35.8 ± 17.6% vs. 4.4 ± 8.7% hepatocytes with adjacent properly localized MDR3 in treated vs. AAV-treated control, respectively; p=2E-5; Fig. 4D).
Inhibition of AAV-specific neutralizing antibodies
AAV-specific neutralizing antibodies (NAbs) are known to be generated following AAV administration. We thus investigated here the impact of the tolerogenic ImmTOR nanoparticles on NAb formation when coadministered with AAV. NAb levels were measured in serum 2, 4, 6, and 8 weeks after the first AAV8-MDR3 administration, with the first samples being taken prior to the second administration. PFIC3 mice administered the vector alone rapidly developed AAV8-specific NAbs after the first administration, which increased after a second administration of the vector up to titers of 1086 ± 399 on average (Fig. 5). In contrast, animals that received ImmTOR in the first injection presented undetectable NAb levels two weeks after administration. These levels slowly increased in two of seven animals that only received AAV vector once together with ImmTOR throughout the experiment albeit at very low levels (final titers: 97 and 105); the remaining 5 animals showed undetectable NAbs. In contrast, AAV8-MDR3+ImmTOR animals that received a second administration of vector developed NAbs afterwards at levels similar to those in animals that received the vector two times without ImmTOR (p=0.94 and 0.98 for 6 and 8 weeks p.i.).
Fig. 5AAV8-specific neutralizing antibodies in infant FVB.Abcb4-/- mice treated with AAV8-MDR3 with and without ImmTOR. Neutralizing antibodies (NAbs) in serum of study animals were quantified via cell-based seroneutralization assay. Lower limit of detection is indicated with a red dotted line. Statistics: 2-way ANOVA, *, p<0.05; **, p<0.01; ***, p<0.001; ***, p<0.0001.
AAV-mediated gene therapy has been developed with the goal of providing durable correction of inherited genetic disorders for which there are no satisfactory therapies. Sustainability of the therapeutic effect still represents a challenge for AAV-mediated gene therapy for pediatric indications involving liver pathology
, as is the case for PFIC3, a devastating monogenic inherited disease only curable by liver transplantation. PFIC3 manifestations occur early in life with one third of patients showing clinical signs of cholestasis within the first year of life
thus, early intervention is necessary to prevent the development of irreversible pathological sequelae like liver cirrhosis. Because it is a monogenic disorder of a gene that is expressed exclusively in the liver
, PFIC3 is a strong candidate for liver-directed AAV-based gene therapy. Previously, we reported a sustained and significant reversal of PFIC3 disease biomarkers following treatment using an AAV vector harboring a codon-optimized sequence of MDR3 isoform A (AAV8-MDR3) in young and adult PFIC3 mice
. However, this strategy has proven to have some important drawbacks. First, MDR3 is not a secreted protein, but a membrane-bound transporter protein, which requires productive transduction of a sufficient percentage of hepatocytes to exceed a threshold in transgene expression necessary to exert a therapeutic benefit. Second, pediatric patients risk experiencing a wane in effect due to the episomal nature of recombinant AAV genomes, which are progressively lost through cell division during liver growth
suggest that this issue may not be restricted only to pediatrics.
Our previous results obtained in the PFIC3 FVB.Abcb4-/- mouse model (in infant pre-fibrotic 2-week- and juvenile fibrotic 5-week-old mice) treated with AAV8-MDR3 showed a significant therapeutic effect; however, several weeks after administration, this effect was lost in a proportion of study animals, pointing to the value of a second administration of the vector. One major hurdle to vector re-administration is the humoral immune response against the first exposure of AAV that results in high titers of NAbs. The formation of NAbs is a host defense mechanism to prevent viral infection, and consequently NAbs are an impediment for AAV-based gene therapy
Effects of transient immunosuppression on adenoassociated, virus-mediated, liver-directed gene transfer in rhesus macaques and implications for human gene therapy.
. For indications that may require repeated treatments, avoidance of this immune response is essential to enable the possibility of readministering AAV gene therapy vectors.
As a method to prevent NAb formation against the therapeutic vector, we tested coadministration with the tolerogenic rapamycin-loaded nanoparticles, or ImmTOR. Two significant observations resulted from the study. First, only AAV8-MDR3 treatment with ImmTOR followed by a second dose of the vector successfully rescued infant PFIC3 mice and provided long-term therapeutic correction of the disease at this dose. The ability to successfully readminister AAV8-MDR3 and to achieve a sustained therapeutic effect correlated with the absence of NAbs two weeks after the first administration (at the time of the second treatment). The degree of the therapeutic correction was striking. The spleen and liver sizes were nearly fully normalized to wild type. There was no increase in serum biomarkers of cholestasis. Liver fibrosis and the appearance of malignant hepatocellular tumors or its precursors were completely prevented. These outcomes were indicative of complete amelioration of disease, and the length of time after treatment that they were observed points to the durability of the therapeutic approach. The disease correction occurred despite only achieving 42% wild-type levels of bile PC. However, previous results also pointed to a therapeutic threshold being substantially less than 100% normalization of wild-type MDR2 functionality
. Second, animals that received ImmTOR with only one dose of AAV8-MDR3 showed improvement, albeit transiently, in cholestatic biomarkers compared to those that were given only AAV8-MDR3. This outcome points to a possible increase in transduction and/or expression when the vector is admixed with ImmTOR, which has been demonstrated elsewhere
. However, due to the nature and the severity of the model, we have consistently observed that once any AAV8-MDR3-mediated therapeutic effect is lost, little to no evidence of transduction or transgene expression is present at sacrifice, especially not when such a long period has transpired since the loss of effect (5-6 months in this instance). Thus, a direct comparison of AAV genome copies or MDR3 expression between the study groups that lost the therapeutic effect in attempts to gauge the effect of ImmTOR on transduction and expression following a single administration is unreliable. Nonetheless, the 1.8 log difference in vector genome copy numbers and MDR3 mRNA transcript levels between treatment with AAV8-MDR3 alone versus a second AAV8-MDR3 after AAV8-MDR3 plus ImmTOR underscores the challenge of treating a rapidly progressing liver disorder in infant animals and the power of being able to re-administer gene therapy.
High titers of NAbs were observed in the sera of the animals that received one or two administrations of the vector alone, which corroborates previous results for similar doses of this vector (data not shown). In contrast, animals treated with ImmTOR at the first treatment instance presented no development of NAbs against the vector capsid at two weeks post-administration, and 5 out of 7 animals treated with a single dose of AAV8-MDR3 with ImmTOR remained seronegative at the final timepoint analyzed, i.e., at 8 weeks, indicative of a sustained tolerogenic effect. However, high NAb titers developed rapidly in animals that received a second dose of the vector in the absence of ImmTOR, indicating that the tolerogenic effect was broken by the high dose of vector. Meliani et al. (2018), in contrast, showed partial blunting of the antibody response to a second, though lower (4x1012 vg/kg), dose of AAV8 vector alone
. Our results differ from those observed combining ImmTOR with protein biologics, such as therapeutic enzymes, coagulation factors, monoclonal antibodies, and recombinant immunotoxin
, wherein immune tolerance was maintained even after multiple challenges of the biologic alone. However, in these cases, immune tolerance was first established by repeated administration of ImmTOR plus antigen. Repeated administration may reinforce immunological memory for immune tolerance, similar to the role of booster immunization for effector immune memory
. In the case of AAV gene therapy, in contrast to other therapies, only a single dose is expected to be administered initially with repeated administrations only coming months or years later, so the opportunity to develop durable immune tolerance capable of withstanding repeated challenge of vector alone may be reduced. Despite this, here we show that the administration of ImmTOR together with AAV8-MDR3 (8x1013 vg/kg) generates a tolerogenic state characterized by the complete inhibition of NAb production against the vector and that this state is enough to enable re-administration of the vector and effective liver transduction. The ability to re-dose in these infant animals was essential to provide a durable increase in transduction and transgene expression and amelioration of disease for at least 8 months, even after the induction of NAb production following the second administration. Nonetheless, given the anticipated low frequency of AAV re-administration in humans, it may be advantageous to coadminister ImmTOR with each dose of AAV gene therapy to maintain the tolerogenic effect with the goal of keeping patients eligible for further redosing if needed
In addition to ameliorating all disease manifestations, the readministration of the vector following co-administration with ImmTOR resulted in the attenuation of the aberrant distribution and proliferation of bile ductular cells, and more importantly in the prevention of the appearance of liver malignancies indicative of HCC. Although the clinical applicability of these findings is lessened due to the fact that HCC is not a manifestation of PFIC3 in humans, it is a valuable finding for the field of AAV-based gene therapy where long-term safety, particularly risk of AAV-mediated insertional mutagenesis, is at the forefront of considerations in translation to the clinic. For example, Dalwadi et al. observed that neonate mice with non-alcoholic fatty liver disease (NAFLD) treated with AAV designed to probe insertional mutagenesis consistently developed HCC, as did a proportion of NAFLD adult mice given a non-targeted control AAV
. Hepatocyte regeneration, due to liver growth or liver injury, was found to be important in the mechanism of AAV-induced oncogenesis. Our findings with repeat AAV treatment in 2-week-old infant mice with a liver damage phenotype and growing livers showed the opposite results, which is highly encouraging. Indeed, the correction of PFIC3 disease via MDR3 gene supplementation and the resulting reduction in hepatocyte proliferation is likely to have played a role in the protection from development of HCC.
To our knowledge, this is the first reported study of ImmTOR in study animals of such a young age. It is possible that the less mature immune system of 2-week-old mice played a role in the outcomes of the study, i.e., that had the study been performed in adult animals the outcomes may have been different. However, the consistent loss of effect in all control animals that did not receive ImmTOR was indicative of an immune response sufficiently capable of inhibiting the second AAV8-MDR3 administration. Whether or not the tolerogenic state observed with ImmTOR arose in part due to the immature immune system of the young mice requires further investigation, although other studies support the ability of ImmTOR to mitigate the immune response to AAV vectors and enable redosing in adult animals
. The conclusion that a second AAV administration was effective only if the first treatment included ImmTOR is a promising sign for the further development of this strategy in pediatric diseases.
Acknowledgments
We would like to thank Dr Céline Bouquet for critical revisions of the manuscript, Dr Carlos Rodríguez-Ortigosa and Sara Arcelus for analysis of serum biomarkers, and Dr Matias A. Avila for help establishing the Abcb4-/- mouse colony. We also would like to thank all personnel at the Animal Facility and Laura Guembe and the Morphology and Image Departments of Cima Universidad de Navarra. IRG was supported financially by a Government of Navarra grant ‘Ayudas para la contratación de doctorandos y doctorandas por empresas y organismos de investigación y difusión de conocimientos “doctorados industriales 2019-2021” expediente: 0011-1408-2018-000016’. Patent pending: WO 2020/074690.
Appendix A. Supplementary data
The following is/are the supplementary data to this article:
Effects of transient immunosuppression on adenoassociated, virus-mediated, liver-directed gene transfer in rhesus macaques and implications for human gene therapy.
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