Ub-ISAP: a streamlined UNIX pipeline for mining unique viral vector integration sites from next generation sequencing data.

BACKGROUND: The analysis of viral vector genomic integration sites is an important component in assessing the safety and efficiency of patient treatment using gene therapy. Alongside this clinical application, integration site identification is a key step in the genetic mapping of viral elements in mutagenesis screens that aim to elucidate gene function. RESULTS: We have developed a UNIX-based vector integration site analysis pipeline (Ub-ISAP) that utilises a UNIX-based workflow for automated integration site identification and annotation of both single and paired-end sequencing reads. Reads that contain viral sequences of interest are selected and aligned to the host genome, and unique integration sites are then classified as transcription start site-proximal, intragenic or intergenic. CONCLUSION: Ub-ISAP provides a reliable and efficient pipeline to generate large datasets for assessing the safety and efficiency of integrating vectors in clinical settings, with broader applications in cancer research. Ub-ISAP is available as an open source software package at https://sourceforge.net/projects/ub-isap/ .

Modeling correction of severe urea cycle defects in the growing murine liver using a hybrid recombinant adeno-associated virus/piggyBac transposase gene delivery system.

UNLABELLED: Liver-targeted gene therapy based on recombinant adeno-associated viral vectors (rAAV) shows promising therapeutic efficacy in animal models and adult-focused clinical trials. This promise, however, is not directly translatable to the growing liver, where high rates of hepatocellular proliferation are accompanied by loss of episomal rAAV genomes and subsequently a loss in therapeutic efficacy. We have developed a hybrid rAAV/piggyBac transposon vector system combining the highly efficient liver-targeting properties of rAAV with stable piggyBac-mediated transposition of the transgene into the hepatocyte genome. Transposition efficiency was first tested using an enhanced green fluorescent protein expression cassette following delivery to newborn wild-type mice, with a 20-fold increase in stably gene-modified hepatocytes observed 4 weeks posttreatment compared to traditional rAAV gene delivery. We next modeled the therapeutic potential of the system in the context of severe urea cycle defects. A single treatment in the perinatal period was sufficient to confer robust and stable phenotype correction in the ornithine transcarbamylase-deficient Spf(ash) mouse and the neonatal lethal argininosuccinate synthetase knockout mouse. Finally, transposon integration patterns were analyzed, revealing 127,386 unique integration sites which conformed to previously published piggyBac data. CONCLUSION: Using a hybrid rAAV/piggyBac transposon vector system, we achieved stable therapeutic protection in two urea cycle defect mouse models; a clinically conceivable early application of this technology in the management of severe urea cycle defects could be as a bridging therapy while awaiting liver transplantation; further improvement of the system will result from the development of highly human liver-tropic capsids, the use of alternative strategies to achieve transient transposase expression, and engineered refinements in the safety profile of piggyBac transposase-mediated integration.

Impact of next-generation sequencing error on analysis of barcoded plasmid libraries of known complexity and sequence.

Barcoded vectors are promising tools for investigating clonal diversity and dynamics in hematopoietic gene therapy. Analysis of clones marked with barcoded vectors requires accurate identification of potentially large numbers of individually rare barcodes, when the exact number, sequence identity and abundance are unknown. This is an inherently challenging application, and the feasibility of using contemporary next-generation sequencing technologies is unresolved. To explore this potential application empirically, without prior assumptions, we sequenced barcode libraries of known complexity. Libraries containing 1, 10 and 100 Sanger-sequenced barcodes were sequenced using an Illumina platform, with a 100-barcode library also sequenced using a SOLiD platform. Libraries containing 1 and 10 barcodes were distinguished from false barcodes generated by sequencing error by a several log-fold difference in abundance. In 100-barcode libraries, however, expected and false barcodes overlapped and could not be resolved by bioinformatic filtering and clustering strategies. In independent sequencing runs multiple false-positive barcodes appeared to be represented at higher abundance than known barcodes, despite their confirmed absence from the original library. Such errors, which potentially impact barcoding studies in an application-dependent manner, are consistent with the existence of both stochastic and systematic error, the mechanism of which is yet to be fully resolved.

Probing the closed-loop model of mRNA translation in living cells.

The mRNA closed-loop, formed through interactions between the cap structure, poly(A) tail, eIF4E, eIF4G and PAB, features centrally in models of eukaryotic translation initiation, although direct support for its existence in vivo is not well established. Here, we investigated the closed-loop using a combination of mRNP isolation from rapidly cross-linked cells and high-throughput qPCR. Using the interaction between these factors and the opposing ends of mRNAs as a proxy for the closed-loop, we provide evidence that it is prevalent for eIF4E/4G-bound but unexpectedly sparse for PAB1-bound mRNAs, suggesting it primarily occurs during a distinct phase of polysome assembly. We observed mRNA-specific variation in the extent of closed-loop formation, consistent with a role for polysome topology in the control of gene expression.

A bioinformatic pipeline for simulating viral integration data.

Viral integration is a complex biological process, and it is useful to have a reference integration dataset with known properties to compare experimental data against, or for comparing with the results from computational tools that detect integration. To generate these data, we developed a pipeline for simulating integrations of a viral or vector genome into a host genome. Our method reproduces more complex characteristics of vector and viral integration, including integration of sub-genomic fragments, structural variation of the integrated genomes, and deletions from the host genome at the integration site. Our method [1] takes the form of a snakemake [2] pipeline, consisting of a Python [3] script using the Biopython [4] module that simulates integrations of a viral reference into a host reference. This produces a reference containing integrations, from which sequencing reads are simulated using ART [5]. The IDs of the reads crossing integration junctions are then annotated using another python script to produce the final output, consisting of the simulated reads and a table of the locations of those integrations and the reads crossing each integration junction. To illustrate our method, we provide simulated reads, integration locations, as well as the code required to simulate integrations using any virus and host reference. This simulation method was used to investigate the performance of viral integration tools in our research [6].

Isling: A Tool for Detecting Integration of Wild-Type Viruses and Clinical Vectors.

Detecting viral and vector integration events is a key step when investigating interactions between viral and host genomes. This is relevant in several fields, including virology, cancer research and gene therapy. For example, investigating integrations of wild-type viruses such as human papillomavirus and hepatitis B virus has proven to be crucial for understanding the role of these integrations in cancer. Furthermore, identifying the extent of vector integration is vital for determining the potential for genotoxicity in gene therapies. To address these questions, we developed isling, the first tool specifically designed for identifying viral integrations in both wild-type and vector from next-generation sequencing data. Isling addresses complexities in integration behaviour including integration of fragmented genomes and integration junctions with ambiguous locations in a host or vector genome, and can also flag possible vector recombinations. We show that isling is up to 1.6-fold faster and up to 170% more accurate than other viral integration tools, and performs well on both simulated and real datasets. Isling is therefore an efficient and application-agnostic tool that will enable a broad range of investigations into viral and vector integration. These include comparisons between integrations of wild-type viruses and gene therapy vectors, as well as assessing the genotoxicity of vectors and understanding the role of viruses in cancer.

Efficient in vivo editing of OTC-deficient patient-derived primary human hepatocytes.

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.

Restoring the natural tropism of AAV2 vectors for human liver.

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.

Attenuation of Heparan Sulfate Proteoglycan Binding Enhances In Vivo Transduction of Human Primary Hepatocytes with AAV2.

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.

Codon-Optimization of Wild-Type Adeno-Associated Virus Capsid Sequences Enhances DNA Family Shuffling while Conserving Functionality.

Adeno-associated virus (AAV) vectors have become one of the most widely used gene transfer tools in human gene therapy. Considerable effort is currently being focused on AAV capsid engineering strategies with the aim of developing novel variants with enhanced tropism for specific human cell types, decreased human seroreactivity, and increased manufacturability. Selection strategies based on directed evolution rely on the generation of highly variable AAV capsid libraries using methods such as DNA-family shuffling, a technique reliant on stretches of high DNA sequence identity between input parental capsid sequences. This identity dependence for reassembly of shuffled capsids is inherently limiting and results in decreased shuffling efficiency as the phylogenetic distance between parental AAV capsids increases. To overcome this limitation, we have developed a novel codon-optimization algorithm that exploits evolutionarily defined codon usage at each amino acid residue in the parental sequences. This method increases average sequence identity between capsids, while enhancing the probability of retaining capsid functionality, and facilitates incorporation of phylogenetically distant serotypes into the DNA-shuffled libraries. This technology will help accelerate the discovery of an increasingly powerful repertoire of AAV capsid variants for cell-type and disease-specific applications.

Identification of liver-specific enhancer-promoter activity in the 3' untranslated region of the wild-type AAV2 genome.

Vectors based on adeno-associated virus type 2 (AAV2) are powerful tools for gene transfer and genome editing applications. The level of interest in this system has recently surged in response to reports of therapeutic efficacy in human clinical trials, most notably for those in patients with hemophilia B (ref. 3). Understandably, a recent report drawing an association between AAV2 integration events and human hepatocellular carcinoma (HCC) has generated controversy about the causal or incidental nature of this association and the implications for AAV vector safety. Here we describe and functionally characterize a previously unknown liver-specific enhancer-promoter element in the wild-type AAV2 genome that is found between the stop codon of the cap gene, which encodes proteins that form the capsid, and the right-hand inverted terminal repeat. This 124-nt sequence is within the 163-nt common insertion region of the AAV genome, which has been implicated in the dysregulation of known HCC driver genes and thus offers added insight into the possible link between AAV integration events and the multifactorial pathogenesis of HCC.

Limiting Thymic Precursor Supply Increases the Risk of Lymphoid Malignancy in Murine X-Linked Severe Combined Immunodeficiency.

In early gene therapy trials for SCID-X1, using γ-retroviral vectors, T cell leukemias developed in a subset of patients secondary to insertional proto-oncogene activation. In contrast, we have reported development of T cell leukemias in SCID-X1 mice following lentivirus-mediated gene therapy independent of insertional mutagenesis. A distinguishing feature in our study was that only a proportion of transplanted γc-deficient progenitors were transduced and therefore competent for reconstitution. We hypothesized that reconstitution of SCID-X1 mice with limiting numbers of hematopoietic progenitors might be a risk factor for lymphoid malignancy. To test this hypothesis, in the absence of transduction, SCID-X1 mice were reconstituted with serially fewer wild-type hematopoietic progenitors. A robust inverse correlation between hematopoietic progenitor cell dose and T-lymphoid malignancy was observed, with earlier disease onset at lower cell doses. Malignancies were of donor origin and carried activating Notch1 mutations. These findings align with emerging evidence that thymocyte self-renewal induced by progenitor deprivation carries an oncogenic risk that is modulated by intra-thymic competition from differentiation-committed cells. Although insertional proto-oncogene activation is required for the development of malignancy in humans, failure of γc-deficient thymocytes to effectively compete with this at-risk cell population may have also contributed to oncogenesis observed in early SCID-X1 trials.

Germline viral "fossils" guide in silico reconstruction of a mid-Cenozoic era marsupial adeno-associated virus.

Germline endogenous viral elements (EVEs) genetically preserve viral nucleotide sequences useful to the study of viral evolution, gene mutation, and the phylogenetic relationships among host organisms. Here, we describe a lineage-specific, adeno-associated virus (AAV)-derived endogenous viral element (mAAV-EVE1) found within the germline of numerous closely related marsupial species. Molecular screening of a marsupial DNA panel indicated that mAAV-EVE1 occurs specifically within the marsupial suborder Macropodiformes (present-day kangaroos, wallabies, and related macropodoids), to the exclusion of other Diprotodontian lineages. Orthologous mAAV-EVE1 locus sequences from sixteen macropodoid species, representing a speciation history spanning an estimated 30 million years, facilitated compilation of an inferred ancestral sequence that recapitulates the genome of an ancient marsupial AAV that circulated among Australian metatherian fauna sometime during the late Eocene to early Oligocene. In silico gene reconstruction and molecular modelling indicate remarkable conservation of viral structure over a geologic timescale. Characterisation of AAV-EVE loci among disparate species affords insight into AAV evolution and, in the case of macropodoid species, may offer an additional genetic basis for assignment of phylogenetic relationships among the Macropodoidea. From an applied perspective, the identified AAV "fossils" provide novel capsid sequences for use in translational research and clinical applications.

Coherence analysis discriminates between retroviral integration patterns in CD34(+) cells transduced under differing clinical trial conditions.

Unequivocal demonstration of the therapeutic utility of γ-retroviral vectors for gene therapy applications targeting the hematopoietic system was accompanied by instances of insertional mutagenesis. These events stimulated the ongoing development of putatively safer integrating vector systems and analysis methods to characterize and compare integration site (IS) biosafety profiles. Continuing advances in next-generation sequencing technologies are driving the generation of ever-more complex IS datasets. Available bioinformatic tools to compare such datasets focus on the association of integration sites (ISs) with selected genomic and epigenetic features, and the choice of these features determines the ability to discriminate between datasets. We describe the scalable application of point-process coherence analysis (CA) to compare patterns produced by vector ISs across genomic intervals, uncoupled from association with genomic features. To explore the utility of CA in the context of an unresolved question, we asked whether the differing transduction conditions used in the initial Paris and London SCID-X1 gene therapy trials result in divergent genome-wide integration profiles. We tested a transduction carried out under each condition, and showed that CA could indeed resolve differences in IS distributions. Existence of these differences was confirmed by the application of established methods to compare integration datasets.