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Wednesday, October 23, 2019

Altering tropism of rAAV by directed evolution.

Directed evolution represents an attractive approach to derive AAV capsid variants capable of selectively infect specific tissue or cell targets. It involves the generation of an initial library of high complexity followed by cycles of selection during which the library is progressively enriched for target-specific variants. Each selection cycle consists of the following: reconstitution of complete AAV genomes within plasmid molecules; production of virions for which each particular capsid variant is matched with the particular capsid gene encoding it; recovery of capsid gene sequences from target tissue after systemic administration. Prevalent variants are then analyzed and evaluated.

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Wednesday, October 23, 2019

AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.

Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and…

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Wednesday, October 23, 2019

Codon swapping of zinc finger nucleases confers expression in primary cells and in vivo from a single lentiviral vector.

Zinc finger nucleases (ZFNs) are promising tools for genome editing for biotechnological as well as therapeutic purposes. Delivery remains a major issue impeding targeted genome modification. Lentiviral vectors are highly efficient for delivering transgenes into cell lines, primary cells and into organs, such as the liver. However, the reverse transcription of lentiviral vectors leads to recombination of homologous sequences, as found between and within ZFN monomers.We used a codon swapping strategy to both drastically disrupt sequence identity between ZFN monomers and to reduce sequence repeats within a monomer sequence. We constructed lentiviral vectors encoding codon-swapped ZFNs or unmodified ZFNs from…

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Wednesday, October 23, 2019

Controlled delivery of ß-globin-targeting TALENs and CRISPR/Cas9 into mammalian cells for genome editing using microinjection.

Tal-effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (Cas) proteins are genome editing tools with unprecedented potential. However, the ability to deliver optimal amounts of these nucleases into mammalian cells with minimal toxicity poses a major challenge. Common delivery approaches are transfection- and viral-based methods; each associated with significant drawbacks. An alternative method for directly delivering genome-editing reagents into single living cells with high efficiency and controlled volume is microinjection. Here, we characterize a glass microcapillary-based injection system and demonstrate controlled co-injection of TALENs or CRISPR/Cas9 together with donor template into single K562 cells for…

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Wednesday, October 23, 2019

Creating and evaluating accurate CRISPR-Cas9 scalpels for genomic surgery.

The simplicity of site-specific genome targeting by type II clustered, regularly interspaced, short palindromic repeat (CRISPR)-Cas9 nucleases, along with their robust activity profile, has changed the landscape of genome editing. These favorable properties have made the CRISPR-Cas9 system the technology of choice for sequence-specific modifications in vertebrate systems. For many applications, whether the focus is on basic science investigations or therapeutic efficacy, activity and precision are important considerations when one is choosing a nuclease platform, target site and delivery method. Here we review recent methods for increasing the activity and accuracy of Cas9 and assessing the extent of off-target cleavage…

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Wednesday, October 23, 2019

Sites of retroviral DNA integration: From basic research to clinical applications.

One of the most crucial steps in the life cycle of a retrovirus is the integration of the viral DNA (vDNA) copy of the RNA genome into the genome of an infected host cell. Integration provides for efficient viral gene expression as well as for the segregation of viral genomes to daughter cells upon cell division. Some integrated viruses are not well expressed, and cells latently infected with human immunodeficiency virus type 1 (HIV-1) can resist the action of potent antiretroviral drugs and remain dormant for decades. Intensive research has been dedicated to understanding the catalytic mechanism of integration, as…

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Wednesday, October 23, 2019

CRISPR/Cas9-generated p47(phox)-deficient cell line for Chronic Granulomatous Disease gene therapy vector development.

Development of gene therapy vectors requires cellular models reflecting the genetic background of a disease thus allowing for robust preclinical vector testing. For human p47(phox)-deficient chronic granulomatous disease (CGD) vector testing we generated a cellular model using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to introduce a GT-dinucleotide deletion (?GT) mutation in p47(phox) encoding NCF1 gene in the human acute myeloid leukemia PLB-985 cell line. CGD is a group of hereditary immunodeficiencies characterized by impaired respiratory burst activity in phagocytes due to a defective phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In Western countries autosomal-recessive p47(phox)-subunit deficiency represents the second…

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Wednesday, October 23, 2019

A high quality assembly of the Nile Tilapia (Oreochromis niloticus) genome reveals the structure of two sex determination regions.

Tilapias are the second most farmed fishes in the world and a sustainable source of food. Like many other fish, tilapias are sexually dimorphic and sex is a commercially important trait in these fish. In this study, we developed a significantly improved assembly of the tilapia genome using the latest genome sequencing methods and show how it improves the characterization of two sex determination regions in two tilapia species.A homozygous clonal XX female Nile tilapia (Oreochromis niloticus) was sequenced to 44X coverage using Pacific Biosciences (PacBio) SMRT sequencing. Dozens of candidate de novo assemblies were generated and an optimal assembly…

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Wednesday, October 23, 2019

Accurate identification and quantification of DNA species by next-generation sequencing in adeno-associated viral vectors produced in insect cells.

Recombinant adeno-associated viral (rAAV) vectors have proven excellent tools for the treatment of many genetic diseases and other complex diseases. However, the illegitimate encapsidation of DNA contaminants within viral particles constitutes a major safety concern for rAAV-based therapies. Moreover, the development of rAAV vectors for early-phase clinical trials has revealed the limited accuracy of the analytical tools used to characterize these new and complex drugs. Although most published data concerning residual DNA in rAAV preparations have been generated by quantitative PCR, we have developed a novel single-strand virus sequencing (SSV-Seq) method for quantification of DNA contaminants in AAV vectors produced…

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Wednesday, October 23, 2019

Short DNA hairpins compromise recombinant adeno-associated virus genome homogeneity.

Short hairpin (sh)RNAs delivered by recombinant adeno-associated viruses (rAAVs) are valuable tools to study gene function in vivo and a promising gene therapy platform. Our data show that incorporation of shRNA transgenes into rAAV constructs reduces vector yield and produces a population of truncated and defective genomes. We demonstrate that sequences with hairpins or hairpin-like structures drive the generation of truncated AAV genomes through a polymerase redirection mechanism during viral genome replication. Our findings reveal the importance of genomic secondary structure when optimizing viral vector designs. We also discovered that shDNAs could be adapted to act as surrogate mutant inverted terminal…

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Wednesday, October 23, 2019

CRISPR/Cas9-mediated scanning for regulatory elements required for HPRT1 expression via thousands of large, programmed genomic deletions.

The extent to which non-coding mutations contribute to Mendelian disease is a major unknown in human genetics. Relatedly, the vast majority of candidate regulatory elements have yet to be functionally validated. Here, we describe a CRISPR-based system that uses pairs of guide RNAs (gRNAs) to program thousands of kilobase-scale deletions that deeply scan across a targeted region in a tiling fashion (“ScanDel”). We applied ScanDel to HPRT1, the housekeeping gene underlying Lesch-Nyhan syndrome, an X-linked recessive disorder. Altogether, we programmed 4,342 overlapping 1 and 2 kb deletions that tiled 206 kb centered on HPRT1 (including 87 kb upstream and 79…

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Wednesday, October 23, 2019

Structural determination of the broadly reactive anti-IGHV1-69 anti-idiotypic antibody G6 and its idiotope.

The heavy chain IGHV1-69 germline gene exhibits a high level of polymorphism and shows biased use in protective antibody (Ab) responses to infections and vaccines. It is also highly expressed in several B cell malignancies and autoimmune diseases. G6 is an anti-idiotypic monoclonal Ab that selectively binds to IGHV1-69 heavy chain germline gene 51p1 alleles that have been implicated in these Ab responses and disease processes. Here, we determine the co-crystal structure of humanized G6 (hG6.3) in complex with anti-influenza hemagglutinin stem-directed broadly neutralizing Ab D80. The core of the hG6.3 idiotope is a continuous string of CDR-H2 residues starting with…

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Wednesday, October 23, 2019

Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses.

Recent advances using CRISPR-Cas9 approaches have dramatically enhanced the ease for genetic manipulation in rodents. Notwithstanding, the methods to deliver nucleic acids into pre-implantation embryos have hardly changed since the original description of mouse transgenesis more than 30 years ago. Here we report a novel strategy to generate genetically modified mice by transduction of CRISPR-Cas9 components into pre-implantation mouse embryos via recombinant adeno-associated viruses (rAAVs). Using this approach, we efficiently generated a variety of targeted mutations in explanted embryos, including indel events produced by non-homologous end joining and tailored mutations using homology-directed repair. We also achieved gene modification in vivo…

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Wednesday, October 23, 2019

Transmission, evolution, and endogenization: Lessons learned from recent retroviral invasions.

Viruses of the subfamily Orthoretrovirinaeare defined by the ability to reverse transcribe an RNA genome into DNA that integrates into the host cell genome during the intracellular virus life cycle. Exogenous retroviruses (XRVs) are horizontally transmitted between host individuals, with disease outcome depending on interactions between the retrovirus and the host organism. When retroviruses infect germ line cells of the host, they may become endogenous retroviruses (ERVs), which are permanent elements in the host germ line that are subject to vertical transmission. These ERVs sometimes remain infectious and can themselves give rise to XRVs. This review integrates recent developments in…

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Wednesday, October 23, 2019

Rapid CRISPR/Cas9-mediated cloning of full-length Epstein-Barr virus genomes from latently infected cells.

Herpesviruses have relatively large DNA genomes of more than 150 kb that are difficult to clone and sequence. Bacterial artificial chromosome (BAC) cloning of herpesvirus genomes is a powerful technique that greatly facilitates whole viral genome sequencing as well as functional characterization of reconstituted viruses. We describe recently invented technologies for rapid BAC cloning of herpesvirus genomes using CRISPR/Cas9-mediated homology-directed repair. We focus on recent BAC cloning techniques of Epstein-Barr virus (EBV) genomes and discuss the possible advantages of a CRISPR/Cas9-mediated strategy comparatively with precedent EBV-BAC cloning strategies. We also describe the design decisions of this technology as well as…

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