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Directed evolution of natural AAV9 using peptide display libraries have been widely used in the search for an optimal recombinant AAV (rAAV) for transgene delivery across the blood-brain barrier (BBB) to the CNS following intravenous ( IV) injection. In this study, we used a different approach by creating a shuffled rAAV capsid library based on parental AAV serotypes 1 through 12. Following selection in mice, 3 novel variants closely related to AAV1, AAV-BBB6, AAV-BBB28, and AAV-BBB31, emerged as top candidates. In direct comparisons with AAV9, our novel variants demonstrated an over 270-fold improvement in CNS transduction and exhibited a clear bias toward neuronal cells. Intriguingly, our AAV-BBB variants relied on the LY6A cellular receptor for CNS entry, similar to AAV9 peptide variants AAV-PHP.eB and AAV.CAP-B10, despite the different bioengineering methods used and parental backgrounds. The variants also showed reduced transduction of both mouse liver and human primary hepatocytes in vivo. To increase clinical translatability, we enhanced the immune escape properties of our new variants by introducing additional modifications based on rational design. Overall, our study highlights the potential of AAV1-like vectors for efficient CNS transduction with reduced liver tropism, offering promising prospects for CNS gene therapies.
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Barrera Hematoencefálica , Terapia Genética , Humanos , Animales , Ratones , Terapia Genética/métodos , Cápside , Hígado , Péptidos/genética , Dependovirus , Vectores Genéticos/genética , Transducción GenéticaRESUMEN
BACKGROUND: Conventional adeno-associated viral (AAV) vectors, while highly effective in quiescent cells such as hepatocytes in the adult liver, confer less durable transgene expression in proliferating cells owing to episome loss. Sustained therapeutic success is therefore less likely in liver disorders requiring early intervention. We have previously developed a hybrid, dual virion approach, recombinant AAV (rAAV)/piggyBac transposon system capable of achieving stable gene transfer in proliferating hepatocytes at levels many fold above conventional AAV vectors. An alternative transposon system, Sleeping Beauty, has been widely used for ex vivo gene delivery; however liver-targeted delivery using a hybrid rAAV/Sleeping Beauty approach remains relatively unexplored. METHODS: We investigated the capacity of a Sleeping Beauty (SB)-based dual rAAV virion approach to achieve stable and efficient gene transfer to the newborn murine liver using transposable therapeutic cassettes encoding coagulation factor IX or ornithine transcarbamylase (OTC). RESULTS: At equivalent doses, rAAV/SB100X transduced hepatocytes with high efficiency, achieving stable expression into adulthood. Compared with conventional AAV, the proportion of hepatocytes transduced, and factor IX and OTC activity levels, were both markedly increased. The proportion of hepatocytes stably transduced increased 4- to 8-fold from <5%, and activity levels increased correspondingly, with markedly increased survival and stable urinary orotate levels in the OTC-deficient Spfash mouse following elimination of residual endogenous murine OTC. CONCLUSIONS: The present study demonstrates the first in vivo utility of a hybrid rAAV/SB100X transposon system to achieve stable long-term therapeutic gene expression following delivery to the highly proliferative newborn mouse liver. These results have relevance to the treatment of genetic metabolic liver diseases with neonatal onset.
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Animales Recién Nacidos , Elementos Transponibles de ADN , Dependovirus , Técnicas de Transferencia de Gen , Vectores Genéticos , Hepatocitos , Hígado , Transducción Genética , Animales , Dependovirus/genética , Elementos Transponibles de ADN/genética , Hígado/metabolismo , Ratones , Vectores Genéticos/genética , Vectores Genéticos/administración & dosificación , Hepatocitos/metabolismo , Factor IX/genética , Ornitina Carbamoiltransferasa/genética , Ornitina Carbamoiltransferasa/metabolismo , Transposasas/genética , Transposasas/metabolismo , Humanos , Transgenes , Terapia Genética/métodos , Ratones Endogámicos C57BLRESUMEN
Recombinant adeno-associated viral (rAAV) vectors are highly promising vehicles for liver-targeted gene transfer, with therapeutic efficacy demonstrated in preclinical models and clinical trials. Progressive familial intrahepatic cholestasis type 3 (PFIC3), an inherited juvenile-onset, cholestatic liver disease caused by homozygous mutation of the ABCB4 gene, may be a promising candidate for rAAV-mediated liver-targeted gene therapy. The Abcb4-/- mice model of PFIC3, with juvenile mice developing progressive cholestatic liver injury due to impaired biliary phosphatidylcholine excretion, resulted in cirrhosis and liver malignancy. Using a conventional rAAV strategy, we observed markedly blunted rAAV transduction in adult Abcb4-/- mice with established liver disease, but not in disease-free, wild-type adults or in homozygous juveniles prior to liver disease onset. However, delivery of predominantly nonintegrating rAAV vectors to juvenile mice results in loss of persistent transgene expression due to hepatocyte proliferation in the growing liver. Conclusion: A hybrid vector system, combining the high transduction efficiency of rAAV with piggyBac transposase-mediated somatic integration, was developed to facilitate stable human ABCB4 expression in vivo and to correct juvenile-onset chronic liver disease in a murine model of PFIC3. A single dose of hybrid vector at birth led to life-long restoration of bile composition, prevention of biliary cirrhosis, and a substantial reduction in tumorigenesis. This powerful hybrid rAAV-piggyBac transposon vector strategy has the capacity to mediate lifelong phenotype correction and reduce the tumorigenicity of progressive familial intrahepatic cholestasis type 3 and, with further refinement, the potential for human clinical translation.
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Subfamilia B de Transportador de Casetes de Unión a ATP/deficiencia , Colestasis Intrahepática/prevención & control , Elementos Transponibles de ADN/genética , Dependovirus/genética , Terapia Genética , Neoplasias Hepáticas Experimentales/prevención & control , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Humanos , Masculino , Ratones , Transducción Genética , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Existing recombinant adeno-associated virus (rAAV) serotypes for delivering in vivo gene therapy treatments for human liver diseases have not yielded combined high-level human hepatocyte transduction and favorable humoral neutralization properties in diverse patient groups. Yet, these combined properties are important for therapeutic efficacy. To bioengineer capsids that exhibit both unique seroreactivity profiles and functionally transduce human hepatocytes at therapeutically relevant levels, we performed multiplexed sequential directed evolution screens using diverse capsid libraries in both primary human hepatocytes in vivo and with pooled human sera from thousands of patients. AAV libraries were subjected to five rounds of in vivo selection in xenografted mice with human livers to isolate an enriched human-hepatotropic library that was then used as input for a sequential on-bead screen against pooled human immunoglobulins. Evolved variants were vectorized and validated against existing hepatotropic serotypes. Two of the evolved AAV serotypes, NP40 and NP59, exhibited dramatically improved functional human hepatocyte transduction in vivo in xenografted mice with human livers, along with favorable human seroreactivity profiles, compared with existing serotypes. These novel capsids represent enhanced vector delivery systems for future human liver gene therapy applications.
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Proteínas de la Cápside/genética , Dependovirus/genética , Ingeniería Genética , Vectores Genéticos/genética , Hígado/metabolismo , Transducción Genética , Animales , Proteínas de la Cápside/química , Femenino , Técnicas de Transferencia de Gen , Hepatocitos/metabolismo , Xenoinjertos , Humanos , Masculino , Ratones , Modelos Moleculares , Conformación ProteicaRESUMEN
The importance of CD4 T cells in tumour immunity has been increasingly recognised, with recent reports describing robust CD4 T cell-dependent tumour control in mice whose immune-regulatory mechanisms have been disturbed by irradiation, chemotherapy, immunomodulatory therapy and/or constitutive immunodeficiency. Tumour control in such models has been attributed in large part to direct Major Histocompatibility Complex (MHC) class II-dependent CD4 T cell killing of tumour cells. To test whether CD4 T cells can eradicate tumours without directly killing tumour cells, we developed an animal model in which tumour-derived antigen could be presented to T-cell receptor (TCR)-transgenic CD4 T cells by host but not tumour MHC class II molecules. In I-E(+) mice bearing I-E(null) tumours, naive I-E-restricted CD4 T cells proliferated locally in tumour-draining lymph nodes after recognising tumour-derived antigen on migratory dendritic cells. In lymphopaenic but not immunosufficient hosts, CD4 T cells differentiated into polarised T helper type 1 (Th1) cells expressing interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα) and interleukin (IL)-2 but little IL-17, and cleared established tumours. Tumour clearance was enhanced by higher TCR affinity for tumour antigen-MHC class II and was critically dependent on IFNγ, as demonstrated by early tumour escape in animals treated with an IFNγ blocking antibody. Thus, CD4 T cells and IFNγ can control tumour growth without direct T-cell killing of tumour cells, and without requiring additional adaptive immune cells such as CD8 T cells and B cells. Our results support a role for effective CD4 T cell-dependent tumour immunity against MHC class II-negative tumours.
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Antígenos de Neoplasias/inmunología , Linfocitos T CD4-Positivos/inmunología , Melanoma/inmunología , Neoplasias Cutáneas/inmunología , Animales , Movimiento Celular , Proliferación Celular , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Interferón gamma/metabolismo , Ganglios Linfáticos/patología , Melanoma/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Neoplasias Cutáneas/patologíaRESUMEN
Despite the availability of life-saving corticosteroids for 70 years, treatment for adrenal insufficiency is not able to recapitulate physiological diurnal cortisol secretion and results in numerous complications. Gene therapy is an attractive possibility for monogenic adrenocortical disorders such as congenital adrenal hyperplasia; however, requires further development of gene transfer/editing technologies and knowledge of the target progenitor cell populations. Vectors based on adeno-associated virus are the leading system for direct in vivo gene delivery but have limitations in targeting replicating cell populations such as in the adrenal cortex. One strategy to overcome this technological limitation is to deliver the relevant adrenocortical gene to a currently targetable organ outside of the adrenal cortex. To explore this possibility, we developed a vector encoding human 21-hydroxylase and directed expression to the liver in a mouse model of congenital adrenal hyperplasia. This extra-adrenal expression resulted in reconstitution of the steroidogenic pathway. Aldosterone and renin levels normalized, and corticosterone levels improved sufficiently to reduce adrenal hyperplasia. This strategy could provide an alternative treatment option for monogenic adrenal disorders, particularly for mineralocorticoid defects. These findings also demonstrate, when targeting the adrenal gland, that inadvertent liver transduction should be precluded as it may confound data interpretation.
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Gene therapies and associated technologies are transforming biomedical research and enabling novel therapeutic options for patients living with debilitating and incurable genetic disorders. The vector system based on recombinant adeno-associated viral vectors (AAVs) has shown great promise in recent clinical trials for genetic diseases of multiple organs, such as the liver and the nervous system. Despite recent successes toward the development of novel bioengineered AAV variants for improved transduction of primary human tissues and cells, vectors that can efficiently transduce human Schwann cells (hSCs) have yet to be identified. Here, we report the application of the functional transduction-RNA selection method in primary hSCs for the development of AAV variants for specific and efficient transgene delivery to hSCs. The two identified capsid variants, Pep2hSC1 and Pep2hSC2, show conserved potency for delivery across various in vitro, in vivo, and ex vivo models of hSCs. These novel AAV capsids will serve as valuable research tools, forming the basis for therapeutic solutions for both SC-related disorders or peripheral nervous system injury.
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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.
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Dependovirus , Vectores Genéticos , Humanos , Vectores Genéticos/genética , Dependovirus/genética , Anticuerpos Neutralizantes , Hígado , PerfusiónRESUMEN
Realization of the immense therapeutic potential of epigenetic editing requires development of clinically predictive model systems that faithfully recapitulate relevant aspects of the target disease pathophysiology. In female patients with ornithine transcarbamylase (OTC) deficiency, an X-linked condition, skewed inactivation of the X chromosome carrying the wild-type OTC allele is associated with increased disease severity. The majority of affected female patients can be managed medically, but a proportion require liver transplantation. With rapid development of epigenetic editing technology, reactivation of silenced wild-type OTC alleles is becoming an increasingly plausible therapeutic approach. Toward this end, privileged access to explanted diseased livers from two affected female infants provided the opportunity to explore whether engraftment and expansion of dissociated patient-derived hepatocytes in the FRG mouse might produce a relevant model for evaluation of epigenetic interventions. Hepatocytes from both infants were successfully used to generate chimeric mouse-human livers, in which clusters of primary human hepatocytes were either OTC positive or negative by immunohistochemistry (IHC), consistent with clonal expansion from individual hepatocytes in which the mutant or wild-type OTC allele was inactivated, respectively. Enumeration of the proportion of OTC-positive or -negative human hepatocyte clusters was consistent with dramatic skewing in one infant and minimal to modest skewing in the other. Importantly, IHC and fluorescence-activated cell sorting analysis of intact and dissociated liver samples from both infants showed qualitatively similar patterns, confirming that the chimeric mouse-human liver model recapitulated the native state in each infant. Also of importance was the induction of a treatable metabolic phenotype, orotic aciduria, in mice, which correlated with the presence of clonally expanded OTC-negative primary human hepatocytes. We are currently using this unique model to explore CRISPR-dCas9-based epigenetic targeting strategies in combination with efficient adeno-associated virus (AAV) gene delivery to reactivate the silenced functional OTC gene on the inactive X chromosome.
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Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa , Ornitina Carbamoiltransferasa , Lactante , Humanos , Ratones , Femenino , Animales , Ornitina Carbamoiltransferasa/genética , Inactivación del Cromosoma X/genética , Hepatocitos , Hígado , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa/genética , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa/terapiaRESUMEN
Recent clinical successes have intensified interest in using adeno-associated virus (AAV) vectors for therapeutic gene delivery. The liver is a key clinical target, given its critical physiological functions and involvement in a wide range of genetic diseases. In the present study, we first investigated the validity of a liver xenograft mouse model repopulated with primary hepatocytes using single-nucleus RNA sequencing (sn-RNA-seq) by studying the transcriptomic profile of human hepatocytes pre- and post-engraftment. Complementary immunofluorescence analyses performed in highly engrafted animals confirmed that the human hepatocytes organize and present appropriate patterns of zone-dependent enzyme expression in this model. Next, we tested a set of rationally designed HSPG de-targeted AAV-LK03 variants for relative transduction performance in human hepatocytes. We used immunofluorescence, next-generation sequencing, and single-nucleus transcriptomics data from highly engrafted FRG mice to demonstrate that the optimally HSPG de-targeted AAV-LK03 displayed a significantly improved lobular transduction profile in this model.
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The liver is a prime target for in vivo gene therapies using recombinant adeno-associated viral vectors. Multiple clinical trials have been undertaken for this target in the past 15 years; however, we are still to see market approval of the first liver-targeted adeno-associated virus (AAV)-based gene therapy. Inefficient expression of the therapeutic transgene, vector-induced liver toxicity and capsid, and/or transgene-mediated immune responses reported at high vector doses are the main challenges to date. One of the contributing factors to the insufficient clinical outcomes, despite highly encouraging preclinical data, is the lack of robust, biologically and clinically predictive preclinical models. To this end, this study reports findings of a functional evaluation of 6 AAV vectors in 12 preclinical models of the human liver, with the aim to uncover which combination of models is the most relevant for the identification of AAV capsid variant for safe and efficient transgene delivery to primary human hepatocytes. The results, generated by studies in models ranging from immortalized cells, iPSC-derived and primary hepatocytes, and primary human hepatic organoids to in vivo models, increased our understanding of the strengths and weaknesses of each system. This should allow the development of novel gene therapies targeting the human liver.
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Dependovirus , Hígado , Humanos , Dependovirus/genética , Hígado/metabolismo , Terapia Genética/métodos , Hepatocitos/metabolismo , Proteínas de la Cápside/metabolismo , Tropismo , Vectores Genéticos/genéticaRESUMEN
The power of adeno-associated viral (AAV)-directed evolution for identifying novel vector variants with improved properties is well established, as evidenced by numerous publications reporting novel AAV variants. However, most capsid variants reported to date have been identified using either replication-competent (RC) selection platforms or polymerase chain reaction-based capsid DNA recovery methods, which can bias the selection toward efficient replication or unproductive intracellular trafficking, respectively. A central objective of this study was to validate a functional transduction (FT)-based method for rapid identification of novel AAV variants based on AAV capsid mRNA expression in target cells. We performed a comparison of the FT platform with existing RC strategies. Based on the selection kinetics and function of novel capsids identified in an in vivo screen in a xenograft model of human hepatocytes, we identified the mRNA-based FT selection as the most optimal AAV selection method. Lastly, to gain insight into the mRNA-based selection mechanism driven by the native AAV-p40 promoter, we studied its activity in a range of in vitro and in vivo targets. We found AAV-p40 to be a ubiquitously active promoter that can be modified for cell-type-specific expression by incorporating binding sites for silencing transcription factors, allowing for cell-type-specific library selection.
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Dependovirus , Vectores Genéticos , Bioingeniería , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Dependovirus/metabolismo , Vectores Genéticos/genética , Humanos , ARN Mensajero , Transducción Genética , TransgenesRESUMEN
Recent clinical successes have intensified interest in using adeno-associated virus (AAV) vectors for therapeutic gene delivery. The liver is a key clinical target, given its critical physiological functions and involvement in a wide range of genetic diseases. Here, we report the bioengineering of a set of next-generation AAV vectors, named AAV-SYDs (where "SYD" stands for Sydney, Australia), with increased human hepato-tropism in a liver xenograft mouse model repopulated with primary human hepatocytes. We followed a two-step process that staggered directed evolution and domain-swapping approaches. Using DNA-family shuffling, we first mapped key AAV capsid regions responsible for efficient human hepatocyte transduction in vivo. Focusing on these regions, we next applied domain-swapping strategies to identify and study key capsid residues that enhance primary human hepatocyte uptake and transgene expression. Our findings underscore the potential of AAV-SYDs as liver gene therapy vectors and provide insights into the mechanism responsible for their enhanced transduction profile.
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Preeclampsia is the leading cause of morbidity and mortality in pregnancy. Although the etiology of preeclampsia is still unclear, it is believed to involve rejection of the fetus, possibly due to an imbalance between regulatory (Treg) and effector T cells. To test this, we compared the frequencies of circulating CD4(+) T cells expressing Foxp3, IFN-gamma, IL-10, or IL-17 at the end of the third trimester of healthy and preeclamptic pregnancies. The size of the Treg cell compartment, defined by the frequency of CD4(+)CD25(high), CD4(+)CD127(low)CD25(+), and CD4(+)Foxp3(+) cells was significantly higher in normal compared with preeclamptic pregnancies. CD4(+)CD25(high) and CD4(+)CD127(low)CD25(+) populations in preeclampsia were not significantly different from those in nonpregnant controls, whereas CD4(+)Foxp3(+) cells numbersre slightly lower in preeclampsia. The suppressive activity of ex vivo-sorted CD4(+)CD127(low)CD25(+) Treg cells was not significantly different between the three study groups. The percentage of CD4(+)IL-17-producing T cells decreased significantly in healthy compared with preeclamptic pregnancies and nonpregnant controls, whereas CD4(+)IL-10- and CD4(+)IFN-gamma-producing cells remained unchanged. Consequently, the ratio of Foxp3(+) Treg to IL-17-expressing CD4(+) T cells was significantly increased in healthy but not in preeclamptic pregnancies. Thus, preeclampsia is associated with the absence of normal systemic skewing away from IL-17 production toward Foxp3(+) expression. Finally, preeclamptic women had significantly higher levels of soluble endoglin, an inhibitor of TGF-beta receptor signaling, which may bias toward IL-17 production. These results suggest that homeostasis between regulatory and proinflammatory CD4(+) T cells might be pivotal for the semiallogeneic fetus to be tolerated within the maternal environment.
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Linfocitos T CD4-Positivos/inmunología , Preeclampsia/inmunología , Embarazo/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Adulto , Antígenos CD/sangre , Linfocitos T CD4-Positivos/metabolismo , Endoglina , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Humanos , Interleucina-17/inmunología , Interleucina-17/metabolismo , Subunidad alfa del Receptor de Interleucina-2/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Subunidad alfa del Receptor de Interleucina-7/inmunología , Subunidad alfa del Receptor de Interleucina-7/metabolismo , Preeclampsia/sangre , Preeclampsia/metabolismo , Receptores de Superficie Celular/sangre , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Reguladores/metabolismoRESUMEN
Recent successes in clinical gene therapy applications have intensified the interest in using adeno-associated viruses (AAVs) as vectors for gene delivery into human liver. An inherent intriguing characteristic of AAVs is that vector variants vary substantially in their ability to transduce hepatocytes from different species. This has historically limited the value of preclinical studies using rodent models for predicting the efficiency of AAV vectors in liver-targeted gene therapy clinical studies. In this work, we aimed to investigate the key determinants of the observed differential interspecies transduction abilities among AAV variants. We took advantage of domain swapping strategies between AAV-KP1, a newly identified variant with enhanced murine liver tropism, and AAV3b, which functions poorly in mice. The systematic in vivo comparison of AAV3b/AAV-KP1 chimeric variants allowed us to identify a threonine insertion at position 265 within variable region I (VR-I) as the key residue that confers murine hepatic transduction to human-derived clade B (AAV2-like) and clade C (AAV3b-like) variants. We propose to use this insertion to generate phylogenetically related AAV surrogates in support of toxicology and dosing studies in the murine liver model.
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BACKGROUND & AIMS: Genome editing technology has immense therapeutic potential and is likely to rapidly supplant contemporary gene addition approaches. Key advantages include the capacity to directly repair mutant loci with resultant recovery of physiological gene expression and maintenance of durable therapeutic effects in replicating cells. In this study, we aimed to repair a disease-causing point mutation in the ornithine transcarbamylase (OTC) locus in patient-derived primary human hepatocytes in vivo at therapeutically relevant levels. METHODS: Editing reagents for precise CRISPR/SaCas9-mediated cleavage and homology-directed repair (HDR) of the human OTC locus were first evaluated against an OTC minigene cassette transposed into the mouse liver. The editing efficacy of these reagents was then tested on the native OTC locus in patient-derived primary human hepatocytes xenografted into the FRG (Fah -/- Rag2 -/- Il2rg -/-) mouse liver. A highly human hepatotropic capsid (NP59) was used for adeno-associated virus (AAV)-mediated gene transfer. Editing events were characterised using next-generation sequencing and restoration of OTC expression was evaluated using immunofluorescence. RESULTS: Following AAV-mediated delivery of editing reagents to patient-derived primary human hepatocytes in vivo, OTC locus-specific cleavage was achieved at efficiencies of up to 72%. Importantly, successful editing was observed in up to 29% of OTC alleles at clinically relevant vector doses. No off-target editing events were observed at the top 10 in silico-predicted sites in the genome. CONCLUSIONS: We report efficient single-nucleotide correction of a disease-causing mutation in the OTC locus in patient-derived primary human hepatocytes in vivo at levels that, if recapitulated in the clinic, would provide benefit for even the most therapeutically challenging liver disorders. Key challenges for clinical translation include the cell cycle dependence of classical HDR and mitigation of unintended on- and off-target editing events. LAY SUMMARY: The ability to efficiently and safely correct disease-causing mutations remains the holy grail of gene therapy. Herein, we demonstrate, for the first time, efficient in vivo correction of a patient-specific disease-causing mutation in the OTC gene in primary human hepatocytes, using therapeutically relevant vector doses. We also highlight the challenges that need to be overcome for this technology to be translated into clinical practice.
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Recent clinical successes in gene therapy applications have intensified interest in using adeno-associated viruses (AAVs) as vectors for therapeutic gene delivery. Although prototypical AAV2 shows robust in vitro transduction of human hepatocyte-derived cell lines, it has not translated into an effective vector for liver-directed gene therapy in vivo. This is consistent with observations made in Fah-/-/Rag2-/-/Il2rg-/- (FRG) mice with humanized livers, showing that AAV2 functions poorly in this xenograft model. Here, we derived naturally hepatotropic AAV capsid sequences from primary human liver samples. We demonstrated that capsid mutations, likely acquired as an unintentional consequence of tissue culture propagation, attenuated the intrinsic human hepatic tropism of natural AAV2 and related human liver AAV isolates. These mutations resulted in amino acid changes that increased binding to heparan sulfate proteoglycan (HSPG), which has been regarded as the primary cellular receptor mediating AAV2 infection of human hepatocytes. Propagation of natural AAV variants in vitro showed tissue culture adaptation with resulting loss of tropism for human hepatocytes. In vivo readaptation of the prototypical AAV2 in FRG mice with a humanized liver resulted in restoration of the intrinsic hepatic tropism of AAV2 through decreased binding to HSPG. Our results challenge the notion that high affinity for HSPG is essential for AAV2 entry into human hepatocytes and suggest that natural AAV capsids of human liver origin are likely to be more effective for liver-targeted gene therapy applications than culture-adapted AAV2.
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Dependovirus , Vectores Genéticos , Animales , Cápside , Dependovirus/genética , Humanos , Hígado , Ratones , Transducción Genética , TropismoRESUMEN
Use of the prototypical adeno-associated virus type 2 (AAV2) capsid delivered unexpectedly modest efficacy in an early liver-targeted gene therapy trial for hemophilia B. This result is consistent with subsequent data generated in chimeric mouse-human livers showing that the AAV2 capsid transduces primary human hepatocytes in vivo with low efficiency. In contrast, novel variants generated by directed evolution in the same model, such as AAV-NP59, transduce primary human hepatocytes with high efficiency. While these empirical data have immense translational implications, the mechanisms underpinning this enhanced AAV capsid transduction performance in primary human hepatocytes are yet to be fully elucidated. Remarkably, AAV-NP59 differs from the prototypical AAV2 capsid by only 11 aa and can serve as a tool to study the correlation between capsid sequence/structure and vector function. Using two orthogonal vectorological approaches, we have determined that just 2 of the 11 changes present in AAV-NP59 (T503A and N596D) account for the enhanced transduction performance of this capsid variant in primary human hepatocytes in vivo, an effect that we have associated with attenuation of heparan sulfate proteoglycan (HSPG) binding affinity. In support of this hypothesis, we have identified, using directed evolution, two additional single amino acid substitution AAV2 variants, N496D and N582S, which are highly functional in vivo. Both substitution mutations reduce AAV2's affinity for HSPG. Finally, we have modulated the ability of AAV8, a highly murine-hepatotropic serotype, to interact with HSPG. The results support our hypothesis that enhanced HSPG binding can negatively affect the in vivo function of otherwise strongly hepatotropic variants and that modulation of the interaction with HSPG is critical to ensure maximum efficiency in vivo. The insights gained through this study can have powerful implications for studies into AAV biology and capsid development for preclinical and clinical applications targeting liver and other organs.
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Adeno-associated virus (AAV) vectors are quickly becoming the vectors of choice for therapeutic gene delivery. To date, hundreds of natural isolates and bioengineered variants have been reported. While factors such as high production titer and low immunoreactivity are important to consider, the ability to deliver the genetic payload (physical transduction) and to drive high transgene expression (functional transduction) remains the most important feature when selecting AAV variants for clinical applications. Reporter expression assays are the most commonly used methods for determining vector fitness. However, such approaches are time consuming and become impractical when evaluating a large number of variants. Limited access to primary human tissues or challenging model systems further complicates vector testing. To address this problem, convenient high-throughput methods based on next-generation sequencing (NGS) are being developed. To this end, we built an AAV Testing Kit that allows inherent flexibility in regard to number and type of AAV variants included, and is compatible with in vitro, ex vivo, and in vivo applications. The Testing Kit presented here consists of a mix of 30 known AAVs where each variant encodes a CMV-eGFP cassette and a unique barcode in the 3'-untranslated region of the eGFP gene, allowing NGS-barcode analysis at both the DNA and RNA/cDNA levels. To validate the AAV Testing Kit, individually packaged barcoded variants were mixed at an equal ratio and used to transduce cells/tissues of interest. DNA and RNA/cDNA were extracted and subsequently analyzed by NGS to determine the physical/functional transduction efficiencies. We were able to assess the transduction efficiencies of immortalized cells, primary cells, and induced pluripotent stem cells in vitro, as well as in vivo transduction in naïve mice and a xenograft liver model. Importantly, while our data validated previously reported transduction characteristics of individual capsids, we also identified novel previously unknown tropisms for some AAV variants.
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Dependovirus/genética , Vectores Genéticos/genética , Ensayos Analíticos de Alto Rendimiento/métodos , Animales , Cápside/metabolismo , Línea Celular , Línea Celular Tumoral , Células Cultivadas , ADN Viral , Femenino , Fibroblastos , Técnicas de Transferencia de Gen , Terapia Genética , Células HeLa , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células Madre Pluripotentes Inducidas , Masculino , Ratones , Receptor EphB2 , Linfocitos T , Transducción Genética , TransgenesRESUMEN
We and others recently described a method for isolating viable forkhead boxp3 (FoxP3(+)) T regulatory cells (Tregs) by means of the surface phenotype CD4(+)CD127(lo)CD25(+). In this study, we used the new strategy to measure Treg numbers, phenotype and function at different ages. Mean percentages of CD4(+)CD127(lo)CD25(+) Tregs increased only slightly throughout life, from 6.10% in cord blood to 7.22% in PBMC from adults between 20 and 25 years and 7.50% in PBMC from adults over the age of 60. In all age groups, a higher proportion of Tregs had acquired a CD45RA(-) memory phenotype compared with CD4(+)Foxp3(-) conventional T cells. This increase was entirely attributable to increased Tregs with an effector memory phenotype, whereas central memory phenotype cells were comparably represented within the Treg and conventional CD4(+) T-cell populations. Expression of CD95 also differed between Tregs and conventional CD4(+) T cells at all ages. However there was no difference in the suppressive capacity of the different naive and memory Treg subsets. These results suggest that, compared with their conventional CD4(+) T-cell counterparts, Tregs undergo preferential differentiation from a naive to an effector memory phenotype, driven by their specificity for self- rather than foreign antigen. However, number and function are remarkably stable throughout life.