RESUMO
Prime editors have high potential for disease modelling and regenerative medicine efforts including those directed at the muscle-wasting disorder Duchenne muscular dystrophy (DMD). However, the large size and multicomponent nature of prime editing systems pose substantial production and delivery issues. Here, we report that packaging optimized full-length prime editing constructs in adenovector particles (AdVPs) permits installing precise DMD edits in human myogenic cells, namely, myoblasts and mesenchymal stem cells (up to 80% and 64%, respectively). AdVP transductions identified optimized prime-editing reagents capable of correcting DMD reading frames of â¼14% of patient genotypes and restoring dystrophin synthesis and dystrophin-ß-dystroglycan linkages in unselected DMD muscle cell populations. AdVPs were equally suitable for correcting DMD iPSC-derived cardiomyocytes and delivering dual prime editors tailored for DMD repair through targeted exon 51 deletion. Moreover, by exploiting the cell cycle-independent AdVP transduction process, we report that 2- and 3-component prime-editing modalities are both most active in cycling than in post-mitotic cells. Finally, we establish that combining AdVP transduction with seamless prime editing allows for stacking chromosomal edits through successive delivery rounds. In conclusion, AdVPs permit versatile investigation of advanced prime editing systems independently of their size and component numbers, which should facilitate their screening and application.
Assuntos
Distrofina , Terapia Genética , Distrofia Muscular de Duchenne , Humanos , Sistemas CRISPR-Cas/genética , Distrofina/genética , Distrofina/metabolismo , Edição de Genes , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Mioblastos/metabolismo , Miócitos Cardíacos/metabolismoRESUMO
Homology-directed recombination (HDR) between donor constructs and acceptor genomic sequences cleaved by programmable nucleases, permits installing large genomic edits in mammalian cells in a precise fashion. Yet, next to precise gene knock-ins, programmable nucleases yield unintended genomic modifications resulting from non-homologous end-joining processes. Alternatively, in trans paired nicking (ITPN) involving tandem single-strand DNA breaks at target loci and exogenous donor constructs by CRISPR-Cas9 nickases, fosters seamless and scarless genome editing. In the present study, we identified high-specificity CRISPR-Cas9 nucleases capable of outperforming parental CRISPR-Cas9 nucleases in directing genome editing through homologous recombination (HR) and homology-mediated end joining (HMEJ) with donor constructs having regular and 'double-cut' designs, respectively. Additionally, we explored the ITPN principle by demonstrating its compatibility with orthogonal and high-specificity CRISPR-Cas9 nickases and, importantly, report that in human induced pluripotent stem cells (iPSCs), in contrast to high-specificity CRISPR-Cas9 nucleases, neither regular nor high-specificity CRISPR-Cas9 nickases activate P53 signaling, a DNA damage-sensing response linked to the emergence of gene-edited cells with tumor-associated mutations. Finally, experiments in human iPSCs revealed that differently from HR and HMEJ genome editing based on high-specificity CRISPR-Cas9 nucleases, ITPN involving high-specificity CRISPR-Cas9 nickases permits editing allelic sequences associated with essentiality and recurrence in the genome.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Humanos , Sistemas CRISPR-Cas/genética , Quebras de DNA de Cadeia Dupla , Edição de Genes/métodos , Genômica , Células-Tronco Pluripotentes Induzidas , MamíferosRESUMO
The electromagnetic field enhancement mechanisms leading to surface-enhanced Raman scattering (SERS) of R6G molecules near Ti3C2TxMXene flakes of different shapes and sizes are analyzed theoretically in this paper. In COMSOL simulations for the enhancement factor (EF) of SERS, the dye molecule is modeled as a small sphere with polarizability spectrum based on experimental data. It is demonstrated, for the first time, that in the wavelength range of500 nm-1000 nm, the enhancement of Raman signals is largely conditioned by quadrupole surface plasmon (QSP) oscillations that induce a strong polarization of the MXene substrate. We show that the vis-NIR spectral range quadrupole SP resonances are strengthened due to interband transitions (IBTs), which provide EF values of the order of 105-107in agreement with experimental data. The weak sensitivity of the EF to the shape and size of MXene nanoparticles (NPs) is interpreted as a consequence of the low dependence of the absorption cross-section of QSP oscillations and IBT on the geometry of the flakes. This reveals a new feature: the independence of EF on the geometry of MXene substrates, which allows to avoid the monitoring of the shape and size of flakes during their synthesis. Thus, MXene flakes can be advantageous for the easy manufacturing of universal substrates for SERS applications. The electromagnetic SERS enhancement is determined by the 'lightning rod' and 'hot-spot' effects due to the partial overlapping of the absorption spectrum of the R6G molecule with these MXene resonances.
RESUMO
Targeted chromosomal insertion of large genetic payloads in human cells leverages and broadens synthetic biology and genetic therapy efforts. Yet, obtaining large-scale gene knock-ins remains particularly challenging especially in hard-to-transfect stem and progenitor cells. Here, fully viral gene-deleted adenovector particles (AdVPs) are investigated as sources of optimized high-specificity CRISPR-Cas9 nucleases and donor DNA constructs tailored for targeted insertion of full-length dystrophin expression units (up to 14.8-kb) through homologous recombination (HR) or homology-mediated end joining (HMEJ). In muscle progenitor cells, donors prone to HMEJ yielded higher CRISPR-Cas9-dependent genome editing frequencies than HR donors, with values ranging between 6% and 34%. In contrast, AdVP transduction of HR and HMEJ substrates in induced pluripotent stem cells (iPSCs) resulted in similar CRISPR-Cas9-dependent genome editing levels. Notably, when compared to regular iPSCs, in p53 knockdown iPSCs, CRISPR-Cas9-dependent genome editing frequencies increased up to 6.7-fold specifically when transducing HMEJ donor constructs. Finally, single DNA molecule analysis by molecular combing confirmed that AdVP-based genome editing achieves long-term complementation of DMD-causing mutations through the site-specific insertion of full-length dystrophin expression units. In conclusion, AdVPs are a robust and flexible platform for installing large genomic edits in human cells and p53 inhibition fosters HMEJ-based genome editing in iPSCs.
Assuntos
Distrofina , Edição de Genes , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Sistemas CRISPR-Cas/genética , Distrofina/genética , Distrofina/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Edição de Genes/métodos , Humanos , Células Musculares/metabolismo , Distrofia Muscular de Duchenne/patologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Prime editing is a recent precision genome editing modality whose versatility offers the prospect for a wide range of applications, including the development of targeted genetic therapies. Yet, an outstanding bottleneck for its optimization and use concerns the difficulty in delivering large prime editing complexes into cells. Here, we demonstrate that packaging prime editing constructs in adenoviral capsids overcomes this constrain resulting in robust genome editing in both transformed and non-transformed human cells with up to 90% efficiencies. Using this cell cycle-independent delivery platform, we found a direct correlation between prime editing activity and cellular replication and disclose that the proportions between accurate prime editing events and unwanted byproducts can be influenced by the target-cell context. Hence, adenovector particles permit the efficacious delivery and testing of prime editing reagents in human cells independently of their transformation and replication statuses. The herein integrated gene delivery and gene editing technologies are expected to aid investigating the potential and limitations of prime editing in numerous experimental settings and, eventually, in ex vivo or in vivo therapeutic contexts.
Assuntos
Adenoviridae/genética , Edição de Genes/métodos , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Deleção de Genes , Células HEK293 , Células HeLa , HumanosRESUMO
RNA-guided nucleases (RGNs) based on CRISPR systems permit installing short and large edits within eukaryotic genomes. However, precise genome editing is often hindered due to nuclease off-target activities and the multiple-copy character of the vast majority of chromosomal sequences. Dual nicking RGNs and high-specificity RGNs both exhibit low off-target activities. Here, we report that high-specificity Cas9 nucleases are convertible into nicking Cas9D10A variants whose precision is superior to that of the commonly used Cas9D10A nickase. Dual nicking RGNs based on a selected group of these Cas9D10A variants can yield gene knockouts and gene knock-ins at frequencies similar to or higher than those achieved by their conventional counterparts. Moreover, high-specificity dual nicking RGNs are capable of distinguishing highly similar sequences by 'tiptoeing' over pre-existing single base-pair polymorphisms. Finally, high-specificity RNA-guided nicking complexes generally preserve genomic integrity, as demonstrated by unbiased genome-wide high-throughput sequencing assays. Thus, in addition to substantially enlarging the Cas9 nickase toolkit, we demonstrate the feasibility in expanding the range and precision of DNA knockout and knock-in procedures. The herein introduced tools and multi-tier high-specificity genome editing strategies might be particularly beneficial whenever predictability and/or safety of genetic manipulations are paramount.
Assuntos
Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Desoxirribonuclease I/metabolismo , Edição de Genes/métodos , Proteínas de Bactérias/genética , Sequência de Bases , Proteína 9 Associada à CRISPR/genética , Células Clonais , Desoxirribonuclease I/genética , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Genes Reporter , Técnicas de Genotipagem , Células HEK293 , Células HeLa , Heterocromatina/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas , Polimorfismo Genético , RNA Guia de Cinetoplastídeos/genética , Proteínas Recombinantes/metabolismo , Streptococcus pyogenes/enzimologia , Especificidade por Substrato , TransfecçãoRESUMO
Genome editing typically involves recombination between donor nucleic acids and acceptor genomic sequences subjected to double-stranded DNA breaks (DSBs) made by programmable nucleases (e.g. CRISPR-Cas9). Yet, nucleases yield off-target mutations and, most pervasively, unpredictable target allele disruptions. Remarkably, to date, the untoward phenotypic consequences of disrupting allelic and non-allelic (e.g. pseudogene) sequences have received scant scrutiny and, crucially, remain to be addressed. Here, we demonstrate that gene-edited cells can lose fitness as a result of DSBs at allelic and non-allelic target sites and report that simultaneous single-stranded DNA break formation at donor and acceptor DNA by CRISPR-Cas9 nickases (in trans paired nicking) mostly overcomes such disruptive genotype-phenotype associations. Moreover, in trans paired nicking gene editing can efficiently and precisely add large DNA segments into essential and multiple-copy genomic sites. As shown herein by genotyping assays and high-throughput genome-wide sequencing of DNA translocations, this is achieved while circumventing most allelic and non-allelic mutations and chromosomal rearrangements characteristic of nuclease-dependent procedures. Our work demonstrates that in trans paired nicking retains target protein dosages in gene-edited cell populations and expands gene editing to chromosomal tracts previously not possible to modify seamlessly due to their recurrence in the genome or essentiality for cell function.
Assuntos
Sistemas CRISPR-Cas/genética , DNA/genética , Desoxirribonuclease I/química , Edição de Genes/métodos , Sequência de Bases , DNA/química , Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Desoxirribonuclease I/genética , Endonucleases/química , Marcação de Genes/métodos , Genoma/genética , Humanos , Mutação/genética , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Genetic therapies based on gene addition have witnessed a variety of clinical successes and the first therapeutic products have been approved for clinical use. Moreover, innovative gene editing techniques are starting to offer new opportunities in which the mutations that underlie genetic diseases can be directly corrected in afflicted somatic cells. The toolboxes underpinning these DNA modifying technologies are expanding with great pace. Concerning the ongoing efforts for their implementation, viral vector-based gene delivery systems have acquired center-stage, providing new hopes for patients with inherited and acquired disorders. Specifically, the application of genetic therapies using viral vectors for the treatment of inborn metabolic disorders is growing and clinical applications are starting to appear. While the field has matured from the technology perspective and has yielded efficacious products, it is the perception of many stakeholders that from the regulatory side further developments are urgently needed. In this review, we summarize the features of state-of-the-art viral vector systems and the corresponding gene-centered therapies they seek to deliver. Moreover, a brief summary is also given on emerging gene editing approaches built on CRISPR-Cas9 nucleases and, more recently, nickases, including base editors and prime editors. Finally, we will point at some regulatory aspects that may deserve further attention for translating these technological developments into actual advanced therapy medicinal products (ATMPs).
Assuntos
Edição de Genes/tendências , Terapia Genética/tendências , Animais , Sistemas CRISPR-Cas , Edição de Genes/métodos , Técnicas de Transferência de Genes , Doenças Genéticas Inatas/terapia , Terapia Genética/métodos , Vetores Genéticos , Humanos , Vírus/genéticaRESUMO
Cranioplasty with titanium mesh provides a stable and cosmetically sound option for the correction of extensive skull bone defects following trauma or tumour surgery with osseous involvement. Meningiomas are for the most part benign lesions that are amenable to surgical cure, however lesions with extradural extension pose additional challenges not only due to increased technical difficulty in achieving gross total resection but also because of distinct biological behaviour. We describe the case of a 43-years-old woman that had been submitted to gross total resection of a WHO grade I falcine and superior sagittal sinus secretory meningioma with extradural and bone extension and cranioplasty with a titanium mesh who had a recurrence 4 years later as two tumour masses on top of the titanium mesh with no adjacent soft tissue invasion, and without dural involvement. To our knowledge, this is the first reported case of meningioma growth on top of titanium cranioplasty material. Seeded or incompletely removed tumoral cells might have exploited the biocompatibility of titanium to promote tumour regrowth.
RESUMO
Enhancing the intracellular delivery and performance of RNA-guided CRISPR-Cas9 nucleases (RGNs) remains in demand. Here, we show that nuclear translocation of commonly used Streptococcus pyogenes Cas9 (SpCas9) proteins is suboptimal. Hence, we generated eCas9.4NLS by endowing the high-specificity eSpCas9(1.1) nuclease (eCas9.2NLS) with additional nuclear localization signals (NLSs). We demonstrate that eCas9.4NLS coupled to prototypic or optimized guide RNAs achieves efficient targeted DNA cleavage and probe the performance of SpCas9 proteins with different NLS compositions at target sequences embedded in heterochromatin versus euchromatin. Moreover, after adenoviral vector (AdV)-mediated transfer of SpCas9 expression units, unbiased quantitative immunofluorescence microscopy revealed 2.3-fold higher eCas9.4NLS nuclear enrichment levels than those observed for high-specificity eCas9.2NLS. This improved nuclear translocation yielded in turn robust gene editing after nonhomologous end joining repair of targeted double-stranded DNA breaks. In particular, AdV delivery of eCas9.4NLS into muscle progenitor cells resulted in significantly higher editing frequencies at defective DMD alleles causing Duchenne muscular dystrophy (DMD) than those achieved by AdVs encoding the parental, eCas9.2NLS, protein. In conclusion, this work provides a strong rationale for integrating viral vector and optimized gene-editing technologies to bring about enhanced RGN delivery and performance.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Terapia Genética , Vetores Genéticos/genética , RNA Guia de Cinetoplastídeos/genéticaRESUMO
BACKGROUND: Facioscapulohumeral dystrophy (FSHD) is associated with partial chromatin relaxation of the DUX4 retrogene containing D4Z4 macrosatellite repeats on chromosome 4, and transcriptional de-repression of DUX4 in skeletal muscle. The common form of FSHD, FSHD1, is caused by a D4Z4 repeat array contraction. The less common form, FSHD2, is generally caused by heterozygous variants in SMCHD1. METHODS: We employed whole exome sequencing combined with Sanger sequencing to screen uncharacterised FSHD2 patients for extra-exonic SMCHD1 mutations. We also used CRISPR-Cas9 genome editing to repair a pathogenic intronic SMCHD1 variant from patient myoblasts. RESULTS: We identified intronic SMCHD1 variants in two FSHD families. In the first family, an intronic variant resulted in partial intron retention and inclusion of the distal 14 nucleotides of intron 13 into the transcript. In the second family, a deep intronic variant in intron 34 resulted in exonisation of 53 nucleotides of intron 34. In both families, the aberrant transcripts are predicted to be non-functional. Deleting the pseudo-exon by CRISPR-Cas9 mediated genome editing in primary and immortalised myoblasts from the index case of the second family restored wild-type SMCHD1 expression to a level that resulted in efficient suppression of DUX4. CONCLUSIONS: The estimated intronic mutation frequency of almost 2% in FSHD2, as exemplified by the two novel intronic SMCHD1 variants identified here, emphasises the importance of screening for intronic variants in SMCHD1. Furthermore, the efficient suppression of DUX4 after restoring SMCHD1 levels by genome editing of the mutant allele provides further guidance for therapeutic strategies.
Assuntos
Proteínas Cromossômicas não Histona/genética , Proteínas de Homeodomínio/genética , Distrofia Muscular Facioescapuloumeral/genética , Adulto , Idoso , Alelos , Sistemas CRISPR-Cas/genética , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Cromossomos Humanos Par 4/genética , Metilação de DNA/genética , Feminino , Edição de Genes/métodos , Expressão Gênica/genética , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Facioescapuloumeral/fisiopatologia , Distrofia Muscular Facioescapuloumeral/terapia , Mutação/genéticaRESUMO
Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disorder caused by mutations in the 2.4 Mb dystrophin-encoding DMD gene. The integration of gene delivery and gene editing technologies based on viral vectors and sequence-specific designer nucleases, respectively, constitutes a potential therapeutic modality for permanently repairing defective DMD alleles in patient-derived myogenic cells. Therefore, we sought to investigate the feasibility of combining adenoviral vectors (AdVs) with CRISPR/Cas9 RNA-guided nucleases (RGNs) alone or together with transcriptional activator-like effector nucleases (TALENs), for endogenous DMD repair through non-homologous end-joining (NHEJ). The strategies tested involved; incorporating small insertions or deletions at out-of-frame sequences for reading frame resetting, splice acceptor knockout for DNA-level exon skipping, and RGN-RGN or RGN-TALEN multiplexing for targeted exon(s) removal. We demonstrate that genome editing based on the activation and recruitment of the NHEJ DNA repair pathway after AdV delivery of designer nuclease genes, is a versatile and robust approach for repairing DMD mutations in bulk populations of patient-derived muscle progenitor cells (up to 37% of corrected DMD templates). These results open up a DNA-level genetic medicine strategy in which viral vector-mediated transient designer nuclease expression leads to permanent and regulated dystrophin synthesis from corrected native DMD alleles.
Assuntos
Distrofina/metabolismo , Endonucleases/metabolismo , Mioblastos/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , Adenoviridae/genética , Alelos , Sequência de Bases , Western Blotting , Sistemas CRISPR-Cas , Linhagem Celular , Reparo do DNA por Junção de Extremidades , Distrofina/genética , Endonucleases/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Células HEK293 , Células HeLa , Humanos , Microscopia de Fluorescência , Dados de Sequência Molecular , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/terapia , Mutação , RNA Guia de Cinetoplastídeos/genética , Transdução GenéticaRESUMO
Transcription activator-like effector nucleases (TALENs) and RNA-guided nucleases derived from clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 systems have become ubiquitous genome editing tools. Despite this, the impact that distinct high-order chromatin conformations have on these sequence-specific designer nucleases is, presently, ill-defined. The same applies to the relative performance of TALENs and CRISPR/Cas9 nucleases at isogenic target sequences subjected to different epigenetic modifications. Here, to address these gaps in our knowledge, we have implemented quantitative cellular systems based on genetic reporters in which the euchromatic and heterochromatic statuses of designer nuclease target sites are stringently controlled by small-molecule drug availability. By using these systems, we demonstrate that TALENs and CRISPR/Cas9 nucleases are both significantly affected by the high-order epigenetic context of their target sequences. In addition, this outcome could also be ascertained for S. pyogenes CRISPR/Cas9 complexes harbouring Cas9 variants whose DNA cleaving specificities are superior to that of the wild-type Cas9 protein. Thus, the herein investigated cellular models will serve as valuable functional readouts for screening and assessing the role of chromatin on designer nucleases based on different platforms or with different architectures or compositions.
Assuntos
Sistemas CRISPR-Cas/genética , Cromatina/genética , Edição de Genes , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Epigênese Genética/genética , Engenharia Genética , Genótipo , Humanos , Conformação Molecular , Streptococcus pyogenes/genéticaRESUMO
Engineered sequence-specific nucleases and donor DNA templates can be customized to edit mammalian genomes via the homologous recombination (HR) pathway. Here we report that the nature of the donor DNA greatly affects the specificity and accuracy of the editing process following site-specific genomic cleavage by transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nucleases. By applying these designer nucleases together with donor DNA delivered as protein-capped adenoviral vector (AdV), free-ended integrase-defective lentiviral vector or nonviral vector templates, we found that the vast majority of AdV-modified human cells underwent scarless homology-directed genome editing. In contrast, a significant proportion of cells exposed to free-ended or to covalently closed HR substrates were subjected to random and illegitimate recombination events. These findings are particularly relevant for genome engineering approaches aiming at high-fidelity genetic modification of human cells.
Assuntos
Adenoviridae/genética , DNA Viral/genética , DNA/química , Desoxirribonucleases/química , Engenharia Genética/métodos , Linhagem Celular , Separação Celular , Marcação de Genes/métodos , Genoma , Células HEK293 , Células HeLa , Humanos , Reação em Cadeia da Polimerase , Recombinação Genética , Sequências Repetitivas de Ácido Nucleico , Reprodutibilidade dos TestesRESUMO
Angle-resolved hemispherical elastic light scattering techniques have been used to reconstruct the surface profile of two-dimensional photonic crystals with submicron resolution and metrological precision. Iterative algorithms permit subsequent calculation of a surface autocorrelation function with additional numerical approximation of the power spectrum and then yield final reconstruction of the surface shape. The proposed method enables filtering out unwanted scattering background, precluding the convergence of phase-retrieval algorithms. The estimation of higher harmonics in the power spectrum provides the reconstruction of a realistic surface achieving subwavelength resolution.
RESUMO
We study the general wave phenomenon of diffractive focusing from a single slit for two types of waves and demonstrate several properties of this effect. Whereas in the first situation, the envelope of a surface gravity water wave is modulated in time by a rectangular function, leading to temporal focusing, in the second example, surface plasmon polariton waves are focused in space by a thin metal slit to a transverse width narrower than the slit itself. The observed evolution of the phase carrier of the water waves is measured for the first time and reveals a nearly flat phase as well as an 80% increase in the intensity at the focal point. We then utilize this flat phase with plasmonic beams in the spatial domain, and study the case of two successive slits, creating a tighter focusing of the waves by putting the second slit at the focal point of the first slit.
RESUMO
Genome editing based on sequence-specific designer nucleases, also known as programmable nucleases, seeks to modify in a targeted and precise manner the genetic information content of living cells. Delivering into cells designer nucleases alone or together with donor DNA templates, which serve as surrogate homologous recombination (HR) substrates, can result in gene knockouts or gene knock-ins, respectively. As engineered replication-defective viruses, viral vectors are having an increasingly important role as delivery vehicles for donor DNA templates and designer nucleases, namely, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated Cas9 (CRISPR-Cas9) nucleases, also known as RNA-guided nucleases (RGNs). We review this dual role played by engineered viral particles on genome editing while focusing on their main scaffolds, consisting of lentiviruses, adeno-associated viruses, and adenoviruses. In addition, the coverage of the growing body of research on the repurposing of viral vectors as delivery systems for genome editing tools is complemented with information regarding their main characteristics, pros, and cons. Finally, this information is framed by a concise description of the chief principles, tools, and applications of the genome editing field as a whole.
Assuntos
Edição de Genes , Engenharia Genética , Vetores Genéticos/genética , Genoma , Vírus/genética , Animais , Endonucleases/genética , Endonucleases/metabolismo , Edição de Genes/métodos , Marcação de Genes , Engenharia Genética/métodos , HumanosRESUMO
Transcription activator-like effector nucleases (TALENs) are designed to cut the genomic DNA at specific chromosomal positions. The resulting DNA double strand break activates cellular repair pathways that can be harnessed for targeted genome modifications. TALENs thus constitute a powerful tool to interrogate the function of DNA sequences within complex genomes. Moreover, their high DNA cleavage activity combined with a low cytotoxicity make them excellent candidates for applications in human gene therapy. Full exploitation of these large and repeat-bearing nucleases in human cell types will benefit largely from using the adenoviral vector (AdV) technology. The genetic stability and the episomal nature of AdV genomes in conjunction with the availability of a large number of AdV serotypes able to transduce various human cell types make it possible to achieve high-level and transient expression of TALENs in numerous target cells, regardless of their mitotic state. Here, we describe a set of protocols detailing the rescue, propagation and purification of TALEN-encoding AdVs. Moreover, we describe procedures for the characterization and quantification of recombinant viral DNA present in the resulting AdV preparations. The protocols are preceded by information about their underlying principles and applied in the context of second-generation capsid-modified AdVs expressing TALENs targeted to the AAVS1 "safe harbor" locus on human chromosome 19.
Assuntos
Adenoviridae/genética , DNA Viral/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , DNA Viral/administração & dosagem , Endonucleases/genética , Vetores Genéticos/administração & dosagem , Células HEK293 , Células HeLa , HumanosRESUMO
The array of genome editing strategies based on targeted double-stranded DNA break formation have recently been enriched through the introduction of transcription activator-like type III effector (TALE) nucleases (TALENs). To advance the testing of TALE-based approaches, it will be crucial to deliver these custom-designed proteins not only into transformed cell types but also into more relevant, chromosomally stable, primary cells. Viral vectors are among the most effective gene transfer vehicles. Here, we investigated the capacity of human immunodeficiency virus type 1- and adenovirus-based vectors to package and deliver functional TALEN genes into various human cell types. To this end, we attempted to assemble particles of these two vector classes, each encoding a monomer of a TALEN pair targeted to a bipartite sequence within the AAVS1 'safe harbor' locus. Vector DNA analyses revealed that adenoviral vectors transferred intact TALEN genes, whereas lentiviral vectors failed to do so, as shown by their heterogeneously sized proviruses in target cells. Importantly, adenoviral vector-mediated TALEN gene delivery resulted in site-specific double-stranded DNA break formation at the intended AAVS1 target site at similarly high levels in both transformed and non-transformed cells. In conclusion, we demonstrate that adenoviral, but not lentiviral, vectors constitute a valuable TALEN gene delivery platform.
Assuntos
Adenoviridae/genética , Proteínas de Bactérias/genética , Desoxirribonucleases/genética , HIV-1/genética , Cromossomos Humanos , Quebras de DNA de Cadeia Dupla , Loci Gênicos , Vetores Genéticos , Células HEK293 , Células HeLa , Humanos , Transdução GenéticaRESUMO
Preclinical and clinical studies showed that autologous transplantation of epidermis derived from genetically modified epithelial stem cells (EpSCs) leads to long-term correction of inherited skin adhesion defects. These studies were based on potentially genotoxic retroviral vectors. We developed an alternative gene transfer strategy aimed at targeting a "safe harbor" locus, the adeno-associated virus integration site 1 (AAVS1), by zinc-finger nuclease (ZFN)-induced homologous recombination (HR). Delivery of AAVS1-specific ZFNs and a GFP-expressing HR cassette by integration-defective lentiviral (LV) vectors (IDLVs) or adenoviral (Ad) vectors resulted in targeted gene addition with an efficiency of > 20% in a human keratinocyte cell line, > 10% in immortalized keratinocytes, and < 1% in primary keratinocytes. Deep sequencing of the AAVS1 locus showed that ZFN-induced double-strand breaks are mostly repaired by nonhomologous end joining (NHEJ) in primary cells, indicating that poor induction of the HR-dependent DNA repair pathway may be a significant limitation for targeted gene integration. Skin equivalents derived from unselected keratinocyte cultures coinfected with a GFP-IDLV and a ZFN-Ad vector were grafted onto immunodeficient mice. GFP-positive clones were observed in all grafts up to 18 weeks post-transplantation. By histological and molecular analysis, we were able to demonstrate highly efficient targeting of the AAVS1 locus in human repopulating EpSCs.