Highlights in PNAS

New for September 12, 2018

Biochemistry

Illustration of Cas9 binding to DNA. Hypothetical PAM sites for Cas9 targets are shown in red; DNA backbones are blue; RNA backbone is teal; DNA and RNA bases are white, except for PAM; histones are green; Cas9 is purple. Image courtesy of Janet Iwasa (University of Utah, Salt Lake City).

Illustration of Cas9 binding to DNA. Hypothetical PAM sites for Cas9 targets are shown in red;
DNA backbones are blue; RNA backbone is teal; DNA and RNA bases are white,
except for PAM; histones are green; Cas9 is purple.
Image courtesy of Janet Iwasa (University of Utah, Salt Lake City).

DNA architecture influences genome-editing efficiency

Biochemical experiments have revealed that the enzyme Cas9, isolated from Streptococcus pyogenes and used in the genome-editing approach CRISPR-Cas9, balks at target DNA sequences assembled into nucleosomes—structures in which DNA is wound around protein spools for packaging into chromosomes. However, whether nucleosomes inhibit Cas9 binding and target DNA cleavage in living cells has been unclear. Using real-time analysis, Robert Yarrington et al. report that Cas9 cleaves target DNA sequences in two gene-regulating stretches—HO and PHO5 promoters—of the yeast genome more efficiently when the targets are located in nucleosome-depleted rather than nucleosome-bound sites. Experimentally evicting nucleosomes through the insertion of binding sites for a nucleosome-displacing protein restored Cas9 target binding and cleavage to levels comparable to naturally nucleosome-depleted sites. Conversely, increasing nucleosome occupancy through mutation of binding sites for a gene switch at the HO locus reduced Cas9 cleavage, suggesting that nucleosomes inhibit Cas9 cleavage. In contrast, zinc-finger nucleases, also common genome-editing tools, appeared largely indifferent to the presence of nucleosomes at target sites. Thus, the authors suggest, nucleosome position maps might help improve genome-editing efficiency in some applications, including human somatic therapies aimed at editing nondividing cells, in which nucleosome positions are relatively static, and base-editing efforts, in which Cas9 may need to sift through nucleosomes to edit target sequences without cleaving them. — P.N.

"Nucleosomes inhibit target cleavage by CRISPR-Cas9 in vivo," by Robert M. Yarrington, Surbhi Verma, Shaina Schwartz, Jonathan K. Trautman, and Dana Carroll
Article 

Immunology and Inflammation; Chemistry

Delivering targeted mRNA cancer vaccines

As a personalized cancer treatment, mRNA vaccines are designed to reprogram patients’ immune systems to recognize tumor-specific antigens and destroy cancerous tissue. However, the approach is currently limited because mRNA does not readily cross cell membranes and degrades rapidly during circulation. Ole Haabeth et al. describe an mRNA vaccine system that uses charge-altering releasable transporters, or CARTs, to deliver customized, functional mRNA to antigen-presenting cells (APCs). Using human peripheral blood mononuclear cells, the authors demonstrate that CART vaccines elicit antigen-specific immune responses to viral epitopes encoded by mRNA. In addition, experiments on a murine model revealed that the CART platform can help treat mice with lymphoma by eliminating medium to large established tumors and conferring protection against subsequent challenges. The study also demonstrates a method to use CARTs to codeliver targeted mRNAs and a Toll-like receptor 9 agonist, which ensures robust activation of APCs. The study uncovers a platform that could aid the development of mRNA cancer vaccines, according to the authors. — T.J.

"mRNA vaccination with charge-altering releasable transporters elicits human T cell responses and cures established tumors in mice," by Ole A. W. Haabeth, et al.
Article

Microbiology

Adenovirus (grey/black) with antibodies (white) inside the cell. The virus is being targeted by TRIM21 (green/blue). Image courtesy of the Laboratory of Molecular Biology, Medical Research Council.

Adenovirus (grey/black) with antibodies (white) inside the cell.
The virus is being targeted by TRIM21 (green/blue).
Image courtesy of the Laboratory of Molecular Biology, Medical Research Council.

Inhibition of transgene expression in adenovirus-vectored gene therapy

Adenoviruses can be manipulated to create relatively safe delivery systems for vaccines, oncological agents, and engineered genes. However, because most humans have been exposed to this common pathogen, preexisting immunity can undercut the effectiveness of adenovirus vectors used in gene therapy. Focusing on adenovirus 5 (Ad5), the viral subtype most commonly adapted as a vector, Maria Bottermann et al. report that anti-Ad5 antibodies in mice inhibit transgene expression in vivo via the cytosolic antibody receptor TRIM21, an intracellular protein that tags invading viruses for destruction. Furthermore, the authors found that TRIM21 blocks the primary mechanism by which Ad5-delivered vaccines confer antiviral and antitumor immunity. The study also includes trials demonstrating that TRIM21 knockout or single antibody mutation restores transgene expression. The results lend insight into the observation that antibodies act as robust inhibitors of transgene expression, despite allowing viral vectors to deliver payloads to a range of targets. According to the authors, the findings highlight the need to develop techniques to augment or disrupt TRIM21 activity. — T.J.

"TRIM21 mediates antibody inhibition of adenovirus-based gene delivery and vaccination," by Maria Bottermann, et al.
Article 

New for September 6, 2018

Medical Sciences

DNA vaccine could aid gene therapy for DMD. Image courtesy of iStock/Natali_Mis.

DNA vaccine could aid gene therapy for DMD. Image courtesy of iStock/Natali_Mis.

DNA vaccine may aid gene therapy for muscular dystrophy

Duchenne muscular dystrophy (DMD) is a potentially fatal, muscle-wasting disorder that afflicts one out of 5,000 male children and stems from genetic defects in the production of dystrophin, a protein found in skeletal and cardiac muscles. Gene therapy trials in which partial dystrophin gene sequences are ferried to muscle cells on an adeno-associated virus (AAV) have shown promise. However, such trials face numerous challenges, including the generation of an immune response in recipients against both the restored dystrophin and AAV vehicle. To circumvent such immune responses, Peggy Ho et al. immunized a mouse model of DMD that lacks dystrophin with a DNA vaccine containing microdystrophin, which is around one-third the size of dystrophin but nonetheless restores some muscle function. One week after gene therapy, the authors injected the vaccine into the quadriceps of mice once weekly for 32 weeks. Gene therapy restored dystrophin production in all mice. Force generation in calf muscles was improved in immunized mice that received gene therapy, compared with mice that received gene therapy but were not immunized. More importantly, mice that received the vaccine for 32 weeks as well as gene therapy exhibited diminished antibody responses against 66 human dystrophin peptides in lab assays, compared with mice that received gene therapy alone. Immunization also appeared to reduce antibody responses against the AAV serotype-6 vehicle used for gene therapy. Follow-up studies over 60 weeks, when several DMD symptoms are heightened, are required to fully assess the vaccine’s efficacy, but the findings suggest a potential approach to improve gene therapy trials for DMD, according to the authors. — P.N.

"Engineered DNA plasmid reduces immunity to dystrophin while improving muscle force in a model of gene therapy of Duchenne dystrophy," by Peggy P. Ho, et al.
Article

Medical Sciences

Phylogenetic framework to assess age of latent HIV reservoirs

The ability of HIV to persist in latent reservoirs is a major barrier to finding a cure, but reconstructing viral reservoir dynamics is challenging. Bradley Jones et al. developed a phylogenetic framework to assess the integration dates of latent HIV lineages within hosts. The authors used plasma HIV RNA sequences sampled before treatment to reconstruct HIV’s within-host evolutionary history, and this information was then used to infer the ages of latent HIV sequences. The authors used the phylogenetic framework to reconstruct HIV sequence ages from a wide variety of within-host datasets, and reconstructed the integration dates of latent HIV sequences sampled from two chronically HIV-infected individuals up to 10 years after the initiation of suppressive therapy. The authors identified and dated reservoir sequences in multiple within-host lineages, with the oldest integration dates occurring up to 21 years before sampling. Genetic bottleneck events, typical of within-host HIV evolution, were also recorded in the reservoir, and sequences from a viremia blip on suppressive therapy suggested that this event was due to spontaneous in vivo reactivation of several ancestral HIV lineages. According to the authors, latent HIV reservoirs can serve as within-host HIV evolutionary archives, and the phylogenetic framework could provide insights into the reservoir dynamics and persistence of HIV. — S.R.

"Phylogenetic approach to recover integration dates of latent HIV sequences within-host," by Bradley R. Jones, et al.
Article 

Neuroscience

Ancient patterns unite halves of mammalian brain

The brain’s left and right hemispheres are connected via the structure known as corpus callosum in placental mammals, or eutherians, and via the structure called anterior commissure in pouched and egg-laying mammals, or marsupials and monotremes. However, whether the pattern of interhemispheric connections in eutherians arose through the evolution of the corpus callosum or instead reflects more ancient principles of mammalian brain organization remains unclear. Rodrigo Suárez et al. compared eutherian datasets with patterns of interhemispheric connections in the monotreme duck-billed platypus and the marsupial fat-tailed dunnart. MRI of fixed dunnart and platypus brains revealed that interhemispheric nerve fibers are spatially arranged within the anterior commissure according to the 3D position of cortical areas, resembling patterns previously observed in the corpus callosum of eutherians. Circuit mapping experiments on dunnart brains revealed additional similarities with known features of the eutherian cortical connectome. According to the authors, the findings provide a framework for future studies on the evolution of vertebrate brain circuits. — J.W.

"A pan-mammalian map of interhemispheric brain connections predates the evolution of the corpus callosum," by Rodrigo Suárez, et al.
Article

Some of the highlights have previously appeared on the PNAS media tipsheet. The articles in PNAS report original research by independent authors and do not necessarily represent the view of the National Academy of Sciences.

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