RESUMO
Enhancers are important cis-regulatory elements controlling cell-type specific expression patterns of genes. Furthermore, combinations of enhancers and minimal promoters are utilized to construct small, artificial promoters for gene delivery vectors. Large-scale functional screening methodology to construct genomic maps of enhancer activities has been successfully established in cultured cell lines, however, not yet applied to terminally differentiated cells and tissues in a living animal. Here, we transposed the Self-Transcribing Active Regulatory Region Sequencing (STARR-seq) technique to the mouse brain using adeno-associated-viruses (AAV) for the delivery of a highly complex screening library tiling entire genomic regions and covering in total 3 Mb of the mouse genome. We identified 483 sequences with enhancer activity, including sequences that were not predicted by DNA accessibility or histone marks. Characterizing the expression patterns of fluorescent reporters controlled by nine candidate sequences, we observed differential expression patterns also in sparse cell types. Together, our study provides an entry point for the unbiased study of enhancer activities in organisms during health and disease.
Assuntos
Elementos Facilitadores Genéticos , Genômica , Animais , Camundongos , Genômica/métodos , Mapeamento Cromossômico/métodos , Regiões Promotoras Genéticas , EncéfaloRESUMO
Display of short peptides on the surface of adeno-associated viruses (AAVs) is a powerful technology for the generation of gene therapy vectors with altered cell specificities and/or transduction efficiencies. Following its extensive prior use in the best characterized AAV serotype 2 (AAV2), recent reports also indicate the potential of other AAV isolates as scaffolds for peptide display. In this study, we systematically explored the respective capacities of 13 different AAV capsid variants to tolerate 27 peptides inserted on the surface followed by production of reporter-encoding vectors. Single-round screening in pre-arrayed 96-well plates permitted rapid and simple identification of superior vectors in >90 cell types, including T cells and primary cells. Notably, vector performance depended not only on the combination of capsid, peptide, and cell type, but also on the position of the inserted peptide and the nature of flanking residues. For optimal data availability and accessibility, all results were assembled in a searchable online database offering multiple output styles. Finally, we established a reverse-transduction pipeline based on vector pre-spotting in 96- or 384-well plates that facilitates high-throughput library panning. Our comprehensive illustration of the vast potential of alternative AAV capsids for peptide display should accelerate their in vivo screening and application as unique gene therapy vectors.
Assuntos
Dependovirus/genética , Peptídeos/metabolismo , Análise Serial de Tecidos/métodos , Terapia Genética , Vetores Genéticos , Humanos , Biblioteca de Peptídeos , Peptídeos/genética , Transdução Genética , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Adeno-associated viruses (AAV) are attractive templates for engineering of synthetic gene delivery vectors. A particularly powerful technology for breeding of novel vectors with improved properties is DNA family shuffling, i.e., generation of chimeric capsids by homology-driven DNA recombination. Here, to make AAV DNA shuffling available to a wider community, we present a robust experimental and bioinformatical pipeline comprising: (i) standardized and partially codon-optimized plasmids carrying 12 different AAV capsid genes; (ii) a scalable protocol including troubleshooting guide for viral library production; and (iii) the freely available software SALANTO for comprehensive analysis of chimeric AAV DNA and protein sequences. Moreover, we describe a set of 12 premade and ready-to-use AAV libraries. Finally, we demonstrate the usefulness of DNA barcoding technology to trace AAV capsid libraries within a complex mixture. Our protocols and resources facilitate the implementation and tailoring of AAV evolution technology in any laboratory interested in customized viral gene transfer.
Assuntos
Dependovirus/genética , Vetores Genéticos/genética , Evolução Molecular , Técnicas de Transferência de GenesRESUMO
Astrocytes, the most abundant cells in the mammalian brain, perform key functions and are involved in several neurodegenerative diseases. The human immunodeficiency virus (HIV) can persist in astrocytes, contributing to the HIV burden and neurological dysfunctions in infected individuals. While a comprehensive approach to HIV cure must include the targeting of HIV-1 in astrocytes, dedicated tools for this purpose are still lacking. Here we report a novel Adeno-associated virus-based vector (AAV9P1) with a synthetic surface peptide for transduction of astrocytes. Analysis of AAV9P1 transduction efficiencies with single brain cell populations, including primary human brain cells, as well as human brain organoids demonstrated that AAV9P1 targeted terminally differentiated human astrocytes much more efficiently than neurons. We then investigated whether AAV9P1 can be used to deliver HIV-inhibitory genes to astrocytes. To this end we generated AAV9P1 vectors containing genes for HIV-1 proviral editing by CRISPR/Cas9. Latently HIV-1 infected astrocytes transduced with these vectors showed significantly diminished reactivation of proviruses, compared with untransduced cultures. Sequence analysis identified mutations/deletions in key HIV-1 transcriptional control regions. We conclude that AAV9P1 is a promising tool for gene delivery to astrocytes and may facilitate inactivation/destruction of persisting HIV-1 proviruses in astrocyte reservoirs.
Assuntos
Astrócitos/fisiologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Dependovirus/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , Vetores Genéticos/administração & dosagem , HIV-1/fisiologia , Astrócitos/efeitos dos fármacos , Astrócitos/virologia , Linhagem Celular Transformada , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/efeitos dos fármacos , Prepúcio do Pênis/citologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Células HEK293 , HIV-1/efeitos dos fármacos , Humanos , MasculinoRESUMO
Liver fibrosis, a form of scarring, develops in chronic liver diseases when hepatocyte regeneration cannot compensate for hepatocyte death. Initially, collagen produced by myofibroblasts (MFs) functions to maintain the integrity of the liver, but excessive collagen accumulation suppresses residual hepatocyte function, leading to liver failure. As a strategy to generate new hepatocytes and limit collagen deposition in the chronically injured liver, we developed in vivo reprogramming of MFs into hepatocytes using adeno-associated virus (AAV) vectors expressing hepatic transcription factors. We first identified the AAV6 capsid as effective in transducing MFs in a mouse model of liver fibrosis. We then showed in lineage-tracing mice that AAV6 vector-mediated in vivo hepatic reprogramming of MFs generates hepatocytes that replicate function and proliferation of primary hepatocytes, and reduces liver fibrosis. Because AAV vectors are already used for liver-directed human gene therapy, our strategy has potential for clinical translation into a therapy for liver fibrosis.
Assuntos
Reprogramação Celular , Dependovirus/genética , Vetores Genéticos/metabolismo , Cirrose Hepática/patologia , Cirrose Hepática/terapia , Fígado/citologia , Miofibroblastos/citologia , Animais , Capsídeo/metabolismo , Proliferação de Células , Técnicas de Transferência de Genes , Camundongos Endogâmicos C57BLRESUMO
Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique combination of beneficial properties(1). These include the apathogenicity of the underlying wildtype viruses and the highly advanced methodologies for production of high-titer, high-purity and clinical-grade recombinant vectors(2). A further particular advantage of the AAV system over other viruses is the availability of a wealth of naturally occurring serotypes which differ in essential properties yet can all be easily engineered as vectors using a common protocol(1,2). Moreover, a number of groups including our own have recently devised strategies to use these natural viruses as templates for the creation of synthetic vectors which either combine the assets of multiple input serotypes, or which enhance the properties of a single isolate. The respective technologies to achieve these goals are either DNA family shuffling(3), i.e. fragmentation of various AAV capsid genes followed by their re-assembly based on partial homologies (typically >80% for most AAV serotypes), or peptide display(4,5), i.e. insertion of usually seven amino acids into an exposed loop of the viral capsid where the peptide ideally mediates re-targeting to a desired cell type. For maximum success, both methods are applied in a high-throughput fashion whereby the protocols are up-scaled to yield libraries of around one million distinct capsid variants. Each clone is then comprised of a unique combination of numerous parental viruses (DNA shuffling approach) or contains a distinctive peptide within the same viral backbone (peptide display approach). The subsequent final step is iterative selection of such a library on target cells in order to enrich for individual capsids fulfilling most or ideally all requirements of the selection process. The latter preferably combines positive pressure, such as growth on a certain cell type of interest, with negative selection, for instance elimination of all capsids reacting with anti-AAV antibodies. This combination increases chances that synthetic capsids surviving the selection match the needs of the given application in a manner that would probably not have been found in any naturally occurring AAV isolate. Here, we focus on the DNA family shuffling method as the theoretically and experimentally more challenging of the two technologies. We describe and demonstrate all essential steps for the generation and selection of shuffled AAV libraries (Fig. 1), and then discuss the pitfalls and critical aspects of the protocols that one needs to be aware of in order to succeed with molecular AAV evolution.
Assuntos
DNA/genética , Dependovirus/genética , Engenharia Genética/métodos , Terapia Genética/métodos , Vetores Genéticos/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Camundongos , Dados de Sequência Molecular , Biblioteca de PeptídeosRESUMO
The human natural killer gene complex (NKC) encodes for numerous C-type lectin-like receptors (CTLR), which are expressed on various immune cells including natural killer (NK) cells and myeloid cells. Certain activation-induced, NKC-encoded CTLR are grouped into the C-type lectin domain family 2 (CLEC2 family) which, in humans, comprises AICL (CLEC2B), CD69 (CLEC2C), and LLT1 (CLEC2D). In this paper, we characterize a novel member of the CLEC2 family, the human orphan gene CLEC2A. The C-type lectin-like domain (CTLD) of CLEC2A is most similar to the CTLD of LLT1 ( approximately 60% similarity). Like mouse CLEC2 family members Clr-b and Clr-g, CLEC2A lacks two highly conserved cysteines (Cys4 and Cys5), which form an intramolecular bond in the CTLD of most CTLR. Alternative splicing of exon 2 and of two distinct terminal exons (exon 5A/B), respectively, gives rise to four CLEC2A variants differing in the usage of the transmembrane domain and/or in the carboxyterminal portion of the CTLD. CLEC2A transcripts were detected primarily in myeloid cell lines, but not in epithelial cell lines. In tissues, CLEC2A is selectively expressed in the skin and, at lower abundance, in hematopoietic and gonadal tissues. Finally, we show that the CLEC2A1 variant is readily expressed at the cell surface, where it may serve as a ligand for NKC-encoded NK receptors.