Harnessing whole human liver ex situ normothermic perfusion for preclinical AAV vector evaluation.

Developing clinically predictive model systems for evaluating gene transfer and gene editing technologies has become increasingly important in the era of personalized medicine. Liver-directed gene therapies present a unique challenge due to the complexity of the human liver. In this work, we describe the application of whole human liver explants in an ex situ normothermic perfusion system to evaluate a set of fourteen natural and bioengineered adeno-associated viral (AAV) vectors directly in human liver, in the presence and absence of neutralizing human sera. Under non-neutralizing conditions, the recently developed AAV variants, AAV-SYD12 and AAV-LK03, emerged as the most functional variants in terms of cellular uptake and transgene expression. However, when assessed in the presence of human plasma containing anti-AAV neutralizing antibodies (NAbs), vectors of human origin, specifically those derived from AAV2/AAV3b, were extensively neutralized, whereas AAV8- derived variants performed efficiently. This study demonstrates the potential of using normothermic liver perfusion as a model for early-stage testing of liver-focused gene therapies. The results offer preliminary insights that could help inform the development of more effective translational strategies.

Topoisomerase Inhibitors Increase Episomal DNA Expression by Inducing the Integration of Episomal DNA in Hepatic Cells.

Gene therapy is a promising strategy to treat and cure most inherited metabolic liver disorders. Viral vectors such as those based on adeno-associated viruses (AAVs) and lentiviruses (LVs) are used as vehicles to deliver functional genes to affected hepatocytes. Adverse events associated with the use of high vector doses have motivated the use of small molecules as adjuvants to reduce the dose. In this study, we showed that a one-hour treatment with topoisomerase inhibitors (camptothecin and etoposide) prior to viral transduction is enough to increase AAV and LV reporter expression in non-dividing hepatic cells in culture. Topoisomerase inhibitors increased both integration-competent (ICLV) and integration-deficient (IDLV) LV-derived expression, with a much stronger increase in the IDLV transduction system. In agreement with that, topoisomerase inhibitors increased viral genome integration in both strains, with a greater impact on the IDLV strain, supporting the idea that topoisomerase inhibitors increased episomal DNA integration, especially when viral integrase activity is abolished. These effects correlated with an increase in the DNA damage response produced by the treatments. Our study highlights the need to monitor DNA damage and undesired integration of viral episomal DNAs into the host genome when studying chemical compounds that increase viral transduction.

Understanding and Tackling Immune Responses to Adeno-Associated Viral Vectors.

As the clinical experience in adeno-associated viral (AAV) vector-based gene therapies is expanding, the necessity to better understand and control the host immune responses is also increasing. Immunogenicity of AAV vectors in humans has been linked to several limitations of the platform, including lack of efficacy due to antibody-mediated neutralization, tissue inflammation, loss of transgene expression, and in some cases, complement activation and acute toxicities. Nevertheless, significant knowledge gaps remain in our understanding of the mechanisms of immune responses to AAV gene therapies, further hampered by the failure of preclinical animal models to recapitulate clinical findings. In this review, we focus on the current knowledge regarding immune responses, spanning from innate immunity to humoral and adaptive responses, triggered by AAV vectors and how they can be mitigated for safer, durable, and more effective gene therapies.

Gene therapy for liver diseases - progress and challenges.

Gene therapy is poised to revolutionize modern medicine, with seemingly unlimited potential for treating and curing genetic disorders. For otherwise incurable indications, including most inherited metabolic liver disorders, gene therapy provides a realistic therapeutic option. In this Review, we discuss gene supplementation and gene editing involving the use of recombinant adeno-associated virus (rAAV) vectors for the treatment of inherited liver diseases, including updates on several ongoing clinical trials that are producing promising results. Clinical testing has been essential in highlighting many key translational challenges associated with this transformative therapy. In particular, the interaction of a patient's immune system with the vector raises issues of safety and the duration of treatment efficacy. Furthermore, several serious adverse events after the administration of high doses of rAAVs suggest greater involvement of innate immune responses and pre-existing hepatic conditions than initially anticipated. Finally, permanent modification of the host genome associated with rAAV genome integration and gene editing raises concerns about the risk of oncogenicity that require careful evaluation. We summarize the main progress, challenges and pathways forward for gene therapy for liver diseases.

High-efficiency purification of divergent AAV serotypes using AAVX affinity chromatography.

The adeno-associated viral vector (AAV) provides a safe and efficient gene therapy platform with several approved products that have marked therapeutic impact for patients. However, a major bottleneck in the development and commercialization of AAV remains the efficiency, cost, and scalability of AAV production. Chromatographic methods have the potential to allow purification at increased scales and lower cost but often require optimization specific to each serotype. Here, we demonstrate that the POROS CaptureSelect AAVX affinity resin efficiently captures a panel of 15 divergent AAV serotypes, including the commonly used AAV2, AAV8, AAV9, PHP.B, and Anc80. We also find that AAVX resin can be regenerated repeatedly without loss of efficiency or carry-over contamination. While AAV preps purified with AAVX showed a higher fraction of empty capsids than preps purified using iodixanol ultracentrifugation, the potency of the AAVX purified vectors was comparable with that of iodixanol purified vectors both in vitro and in vivo. Finally, optimization of the purification protocol resulted in a process with an overall efficiency of 65%-80% across all scales and AAV serotypes tested. These data establish AAVX affinity chromatography as a versatile and efficient method for purification of a broad range of AAV serotypes.

Ancestral library identifies conserved reprogrammable liver motif on AAV capsid.

Gene therapy is emerging as a modality in 21st-century medicine. Adeno-associated viral (AAV) gene transfer is a leading technology to achieve efficient and durable expression of a therapeutic transgene. However, the structural complexity of the capsid has constrained efforts to engineer the particle toward improved clinical safety and efficacy. Here, we generate a curated library of barcoded AAVs with mutations across a variety of functionally relevant motifs. We then screen this library in vitro and in vivo in mice and nonhuman primates, enabling a broad, multiparametric assessment of every vector within the library. Among the results, we note a single residue that modulates liver transduction across all interrogated models while preserving transduction in heart and skeletal muscles. Moreover, we find that this mutation can be grafted into AAV9 and leads to profound liver detargeting while retaining muscle transduction-a finding potentially relevant to preventing hepatoxicities seen in clinical studies.

Choice of vector and surgical approach enables efficient cochlear gene transfer in nonhuman primate.

Inner ear gene therapy using adeno-associated viral vectors (AAV) promises to alleviate hearing and balance disorders. We previously established the benefits of Anc80L65 in targeting inner and outer hair cells in newborn mice. To accelerate translation to humans, we now report the feasibility and efficiency of the surgical approach and vector delivery in a nonhuman primate model. Five rhesus macaques were injected with AAV1 or Anc80L65 expressing eGFP using a transmastoid posterior tympanotomy approach to access the round window membrane after making a small fenestra in the oval window. The procedure was well tolerated. All but one animal showed cochlear eGFP expression 7-14 days following injection. Anc80L65 in 2 animals transduced up to 90% of apical inner hair cells; AAV1 was markedly less efficient at equal dose. Transduction for both vectors declined from apex to base. These data motivate future translational studies to evaluate gene therapy for human hearing disorders.

Pharmacological Induction of a Progenitor State for the Efficient Expansion of Primary Human Hepatocytes.

The liver is an organ with strong regenerative capacity, yet primary hepatocytes have a low amplification potential in vitro, a major limitation for the cell-based therapy of liver disorders and for ex vivo biological screens. Induced pluripotent stem cells (iPSCs) may help to circumvent this obstacle but often harbor genetic and epigenetic abnormalities, limiting their potential. Here, we describe the pharmacological induction of proliferative human hepatic progenitor cells (HPCs) through a cocktail of growth factors and small molecules mimicking the signaling events involved in liver regeneration. Human HPCs from healthy donors and pediatric patients proliferated vigorously while maintaining their genomic stability and could be redifferentiated in vitro into metabolically competent cells that supported the replication of hepatitis B and delta viruses. Redifferentiation efficiency was boosted by three-dimensional culture. Finally, transcriptome analysis showed that HPCs were more closely related to mature hepatocytes than iPSC-derived hepatocyte-like cells were. Conclusion: HPC induction holds promise for a variety of applications such as ex vivo disease modeling, personalized drug testing or metabolic studies, and development of a bioartificial liver.

Human Hepatocyte-Derived Induced Pluripotent Stem Cells: MYC Expression, Similarities to Human Germ Cell Tumors, and Safety Issues.

Induced pluripotent stem cells (iPSC) are a most promising approach to the development of a hepatocyte transplantable mass sufficient to induce long-term correction of inherited liver metabolic diseases, thus avoiding liver transplantation. Their intrinsic self-renewal ability and potential to differentiate into any of the three germ layers identify iPSC as the most promising cell-based therapeutics, but also as drivers of tumor development. Teratoma development currently represents the gold standard to assess iPSC pluripotency. We analyzed the tumorigenic potential of iPSC generated from human hepatocytes (HEP-iPSC) and compared their immunohistochemical profiles to that of tumors developed from fibroblast and hematopoietic stem cell-derived iPSC. HEP-iPSC generated tumors significantly presented more malignant morphological features than reprogrammed fibroblasts or CD34+ iPSC. Moreover, the protooncogene myc showed the strongest expression in HEP-iPSC, compared to only faint expression in the other cell subsets. Random integration of transgenes and the use of potent protooncogenes such as myc might be a risk factor for malignant tumor development if hepatocytes are used for reprogramming. Nonviral vector delivery systems or reprogramming of cells obtained from less invasive harvesting methods would represent interesting options for future developments in stem cell-based approaches for liver metabolic diseases.

Loss of transcriptional control over endogenous retroelements during reprogramming to pluripotency.

Endogenous retroelements (EREs) account for about half of the mouse or human genome, and their potential as insertional mutagens and transcriptional perturbators is suppressed by early embryonic epigenetic silencing. Here, we asked how ERE control is maintained during the generation of induced pluripotent stem cells (iPSCs), as this procedure involves profound epigenetic remodeling. We found that all EREs tested were markedly up-regulated during the reprogramming of either mouse embryonic fibroblasts, human CD34(+) cells, or human primary hepatocytes. At the iPSC stage, EREs of some classes were repressed, whereas others remained highly expressed, yielding a pattern somewhat reminiscent of that recorded in embryonic stem cells. However, variability persisted between individual iPSC clones in the control of specific ERE integrants. Both during reprogramming and in iPS cells, the up-regulation of specific EREs significantly impacted on the transcription of nearby cellular genes. While transcription triggered by specific ERE integrants at highly precise developmental stages may be an essential step toward obtaining pluripotent cells, the broad and unspecific unleashing of the repetitive genome observed here may contribute to the inefficiency of the reprogramming process and to the phenotypic heterogeneity of iPSCs.

Innate functions of immunoglobulin M lessen liver gene transfer with helper-dependent adenovirus.

The immune system poses obstacles to viral vectors, even in the first administration to preimmunized hosts. We have observed that the livers of B cell-deficient mice were more effectively transduced by a helper-dependent adenovirus serotype-5 (HDA) vector than those of WT mice. This effect was T-cell independent as shown in athymic mice. Passive transfer of the serum from adenovirus-naïve WT to Rag1KO mice resulted in a reduction in gene transfer that was traced to IgM purified from serum of adenovirus-naïve mice. To ascribe the gene transfer inhibition activity to either adenoviral antigen-specific or antigen-unspecific functions of IgM, we used a monoclonal IgM antibody of unrelated specificity. Both the polyclonal and the irrelevant monoclonal IgM inhibited gene transfer by the HDA vector to either cultured hepatocellular carcinoma cells or to the liver of mice in vivo. Adsorption of polyclonal or monoclonal IgMs to viral capsids was revealed by ELISAs on adenovirus-coated plates. These observations indicate the existence of an inborn IgM mechanism deployed against a prevalent virus to reduce early post-infection viremia. In conclusion, innate IgM binding to adenovirus serotype-5 capsids restrains gene-transfer and offers a mechanism to be targeted for optimization of vector dosage in gene therapy with HDA vectors.

Safety and liver transduction efficacy of rAAV5-cohPBGD in nonhuman primates: a potential therapy for acute intermittent porphyria.

Acute intermittent porphyria (AIP) results from haplo-insufficient activity of porphobilinogen deaminase (PBGD) and is characterized clinically by life-threatening, acute neurovisceral attacks. To date, liver transplantation is the only curative option for AIP. The aim of the present preclinical nonhuman primate study was to determine the safety and transduction efficacy of an adeno-associated viral vector encoding PBGD (recombinant AAV serotype 5-codon-optimized human porphobilinogen deaminase, rAAV5-cohPBGD) administered intravenously as part of a safety program to start a clinical study in patients with AIP. Macaques injected with either 1 × 10(13) or 5 × 10(13) vector genomes/kg of clinical-grade rAAV5-cohPBGD were monitored by standardized clinical parameters, and vector shedding was analyzed. Liver transduction efficacy, biodistribution, vector integration, and histopathology at day 30 postvector administration were determined. There was no evidence of acute toxicity, and no adverse effects were observed. The vector achieved efficient and homogenous hepatocellular transduction, reaching transgenic PBGD expression levels equivalent to 50% of the naturally expressed PBGD mRNA. No cellular immune response was detected against the human PBGD or AAV capsid proteins. Integration site analysis in transduced liver cells revealed an almost random integration pattern supporting the good safety profile of rAAV5-cohPBGD. Together, data obtained in nonhuman primates indicate that rAAV5-cohPBGD represents a safe therapy to correct the metabolic defect present in AIP patients.

Helper-dependent adenoviral liver gene therapy protects against induced attacks and corrects protein folding stress in acute intermittent porphyria mice.

Acute intermittent porphyria (AIP) is a hepatic metabolic disease that results from haplo-insufficient activity of porphobilinogen deaminase (PBGD). The dominant clinical feature is acute intermittent attacks when hepatic heme synthesis is activated by endocrine or exogenous factors. Gene therapy vectors over-expressing PBGD protein in the liver offers potential as a cure for AIP. Here, we developed a helper-dependent adenovirus (HDA) encoding human PBGD (hPBGD) and assessed its therapeutic efficacy in a murine model of AIP. Intravenous or intrahepatic administration of HDA-hPBGD to AIP mice resulted in a sustained hepatic hPBGD expression in a dose-dependent manner. Intrahepatic administration conveyed full protection against induced porphyria attacks at a significantly lower viral dose than intravenous injection. Transgenic hPBGD accumulated only in the cytosol of hepatocytes as the endogenous protein. Characterization of PBGD-deficient mouse strains revealed that a strong PBGD deficiency causes the chronic disturbance of cytosolic and endoplasmic reticulum folding machineries. This disturbance was completely restored over time by the over-expression of hPBGD. HDA-hPBGD is a promising vector that protects against porphyria attacks and resolves the chronic folding stress associated with low levels of PBGD activity.

Transient and intensive pharmacological immunosuppression fails to improve AAV-based liver gene transfer in non-human primates.

BACKGROUND: Adeno-associated vectors (rAAV) have been used to attain long-term liver gene expression. In humans, the cellular immune response poses a serious obstacle for transgene persistence while neutralizing humoral immunity curtails re-administration. Porphobilinogen deaminase (PBGD) haploinsufficiency (acute intermittent porphyria) benefits from liver gene transfer in mouse models and clinical trials are about to begin. In this work, we sought to study in non-human primates the feasibility of repeated gene-transfer with intravenous administration of rAAV5 vectors under the effects of an intensive immunosuppressive regimen and to analyze its ability to circumvent T-cell immunity and thereby prolong transgene expression. METHODS: Three female Macaca fascicularis were intravenously injected with 1 x 10(13) genome copies/kg of rAAV5 encoding the human PBGD. Mycophenolate mofetil (MMF), anti-thymocyte immunoglobulin, methylprednisolone, tacrolimus and rituximab were given in combination during 12 weeks to block T- and B-cell mediated adaptive immune responses in two macaques. Immunodeficient and immunocompetent mice were intravenously injected with 5 x 10(12) genome copies/kg of rAAV5-encoding luciferase protein. Forty days later MMF, tacrolimus and rituximab were daily administrated to ascertain whether the immunosuppressants or their metabolites could interfere with transgene expression. RESULTS: Macaques given a rAAV5 vector encoding human PBGD developed cellular and humoral immunity against viral capsids but not towards the transgene. Anti-AAV humoral responses were attenuated during 12 weeks but intensely rebounded following cessation of the immunosuppressants. Accordingly, subsequent gene transfer with a rAAV5 vector encoding green fluorescent protein was impossible. One macaque showed enhanced PBGD expression 25 weeks after rAAV5-pbgd administration but overexpression had not been detected while the animal was under immunosuppression. As a potential explanation, MMF decreases transgene expression in mouse livers that had been successfully transduced by a rAAV5 several weeks before MMF onset. Such a silencing effect was independent of AAV complementary strand synthesis and requires an adaptive immune system. CONCLUSIONS: These results indicate that our transient and intensive pharmacological immunosuppression fails to improve AAV5-based liver gene transfer in non-human primates. The reasons include an incomplete restraint of humoral immune responses to viral capsids that interfere with repeated gene transfer in addition to an intriguing MMF-dependent drug-mediated interference with liver transgene expression.

Renal failure affects the enzymatic activities of the three first steps in hepatic heme biosynthesis in the acute intermittent porphyria mouse.

Chronic kidney disease is a long-term complication in acute intermittent porphyria (AIP). The pathophysiological significance of hepatic overproduction of the porphyrin precursors aminolevulinate acid (ALA) and porphobilinogen (PBG) in chronic kidney disease is unclear. We have investigated the effect of repetitive acute attacks on renal function and the effect of total or five-sixth nephrectomy causing renal insufficiency on hepatic heme synthesis in the porphobilinogen deaminase (PBGD)-deficient (AIP) mouse. Phenobarbital challenge in the AIP-mice increased urinary porphyrin precursor excretion. Successive attacks throughout 14 weeks led to minor renal lesions with no impact on renal function. In the liver of wild type and AIP mice, 5/6 nephrectomy enhanced transcription of the first and rate-limiting ALA synthase. As a consequence, urinary PBG excretion increased in AIP mice. The PBG/ALA ratio increased from 1 in sham operated AIP animals to over 5 (males) and over 13 (females) in the 5/6 nephrectomized mice. Total nephrectomy caused a rapid decrease in PBGD activity without changes in enzyme protein level in the AIP mice but not in the wild type animals. In conclusion, high concentration of porphyrin precursors had little impact on renal function. However, progressive renal insufficiency aggravates porphyria attacks and increases the PBG/ALA ratio, which should be considered a warning sign for potentially life-threatening impairment in AIP patients with signs of renal failure.

Sustained enzymatic correction by rAAV-mediated liver gene therapy protects against induced motor neuropathy in acute porphyria mice.

Acute intermittent porphyria (AIP) is characterized by a hereditary deficiency of hepatic porphobilinogen deaminase (PBGD) activity. Clinical features are acute neurovisceral attacks accompanied by overproduction of porphyrin precursors in the liver. Recurrent life-threatening attacks can be cured only by liver transplantation. We developed recombinant adeno-associated virus (rAAV) vectors expressing human PBGD protein driven by a liver-specific promoter to provide sustained protection against induced attacks in a predictive model for AIP. Phenobarbital injections in AIP mice induced porphyrin precursor accumulation, functional block of nerve conduction, and progressive loss of large-caliber axons in the sciatic nerve. Hepatocyte transduction showed no gender variation after rAAV2/8 injection, while rAAV2/5 showed lower transduction efficiency in females than males. Full protection against induced phenobarbital-attacks was achieved in animals showing over 10% of hepatocytes expressing high amounts of PBGD. More importantly, sustained hepatic expression of hPBGD protected against loss of large-caliber axons in the sciatic nerve and disturbances in nerve conduction velocity as induced by recurrent phenobarbital administrations. These data show for the first time that porphyrin precursors generated in the liver interfere with motor function. rAAV2/5-hPBGD vector can be produced in sufficient quantity for an intended gene therapy trial in patients with recurrent life-threatening porphyria attacks.

Intensive pharmacological immunosuppression allows for repetitive liver gene transfer with recombinant adenovirus in nonhuman primates.

Repeated administration of gene therapies is hampered by host immunity toward vectors and transgenes. Attempts to circumvent antivector immunity include pharmacological immunosuppression or alternating different vectors and vector serotypes with the same transgene. Our studies show that B-cell depletion with anti-CD20 monoclonal antibody and concomitant T-cell inhibition with clinically available drugs permits repeated liver gene transfer to a limited number of nonhuman primates with recombinant adenovirus. Adenoviral vector-mediated transfer of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene was visualized in vivo with a semiquantitative transgene-specific positron emission tomography (PET) technique, liver immunohistochemistry, and immunoblot for the reporter transgene in needle biopsies. Neutralizing antibody and T cell-mediated responses toward the viral capsids were sequentially monitored and found to be repressed by the drug combinations tested. Repeated liver transfer of the HSV1-tk reporter gene with the same recombinant adenoviral vector was achieved in macaques undergoing a clinically feasible immunosuppressive treatment that ablated humoral and cellular immune responses. This strategy allows measurable gene retransfer to the liver as late as 15 months following the first adenoviral exposure in a macaque, which has undergone a total of four treatments with the same adenoviral vector.

Porphobilinogen deaminase over-expression in hepatocytes, but not in erythrocytes, prevents accumulation of toxic porphyrin precursors in a mouse model of acute intermittent porphyria.

BACKGROUND & AIMS: Acute intermittent porphyria (AIP) is characterized by hepatic porphobilinogen deaminase (PBGD) deficiency resulting in a marked overproduction of presumably toxic porphyrin precursors. Our study aimed to assess the protective effects of bone marrow transplantation or PBGD gene transfer into the liver against phenotypic manifestations of acute porphyria attack induced in an AIP murine model. METHODS: Lethally irradiated AIP mice were intravenously injected with 5x10(6) nucleated bone marrow cells from wild type or AIP donor mice. To achieve liver gene transfer, AIP mice received via hydrodynamic injection plasmids expressing human PBGD or luciferase, driven by a liver-specific promoter. RESULTS: Erythrocyte PBGD activity increased 2.4-fold in AIP mice receiving bone marrow cells from normal animals. Nevertheless, phenobarbital administration in these mice reproduced key features of acute attacks, such as massively increased urinary porphyrin precursor excretion and decreased motor coordination. Hepatic PBGD activity increased 2.2-fold after hydrodynamic injection of therapeutic plasmid. Mice injected with the luciferase control plasmid showed a high excretion of porphyrin precursors after phenobarbital administration whereas just a small increase was observed in AIP mice injected with the PBGD plasmid. Furthermore, motor disturbance was almost completely abolished in AIP mice treated with the therapeutic plasmid. CONCLUSIONS: PBGD deficiency in erythroid tissue is not associated with phenotypic manifestations of acute porphyria. In contrast, PBGD over-expression in hepatocytes, albeit in a low proportion, reduced precursor accumulation, which is the hallmark of acute porphyric attacks. Liver-directed gene therapy might offer an alternative to liver transplantation applicable in patients with severe and recurrent manifestations.