High-resolution epitope mapping by AllerScan reveals relationships between IgE and IgG repertoires during peanut oral immunotherapy.

Peanut allergy can result in life-threatening reactions and is a major public health concern. Oral immunotherapy (OIT) induces desensitization to food allergens through administration of increasing amounts of allergen. To dissect peanut-specific immunoglobulin E (IgE) and IgG responses in subjects undergoing OIT, we have developed AllerScan, a method that leverages phage-display and next-generation sequencing to identify the epitope targets of peanut-specific antibodies. We observe a striking diversification and boosting of the peanut-specific IgG repertoire after OIT and a reduction in pre-existing IgE levels against individual epitopes. High-resolution epitope mapping reveals shared recognition of public epitopes in Ara h 1, 2, 3, and 7. In individual subjects, OIT-induced IgG specificities overlap extensively with IgE and exhibit strikingly similar antibody footprints, suggesting related clonal lineages or convergent evolution of peanut-specific IgE and IgG B cells. Individual differences in epitope recognition identified via AllerScan could inform safer and more effective personalized immunotherapy.

Author Correction: Immune-orthogonal orthologues of AAV capsids and of Cas9 circumvent the immune response to the administration of gene therapy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Immune-orthogonal orthologues of AAV capsids and of Cas9 circumvent the immune response to the administration of gene therapy.

Protein-based therapeutics can activate the adaptive immune system, leading to the production of neutralizing antibodies and the clearance of the treated cells mediated by cytotoxic T cells. Here, we show that the sequential use of immune-orthogonal orthologues of CRISPR-associated protein 9 (Cas9) and adeno-associated viruses (AAVs) evades adaptive immune responses and enables effective gene editing using repeated dosing. We compared total sequence similarities and predicted binding strengths to class-I and class-II major histocompatibility complex (MHC) proteins for 284 DNA-targeting and 84 RNA-targeting CRISPR effectors and 167 AAV VP1-capsid-protein orthologues. We predict the absence of cross-reactive immune responses for 79% of the DNA-targeting Cas orthologues-which we validated for three Cas9 orthologues in mice-yet we anticipate broad immune cross-reactivity among the AAV serotypes. We also show that efficacious in vivo gene editing is uncompromised when using multiple dosing with orthologues of AAVs and Cas9 in mice that were previously immunized against the AAV vector and the Cas9 cargo. Multiple dosing with protein orthologues may allow for sequential regimens of protein therapeutics that circumvent pre-existing immunity or induced immunity.

RNA-Guided Adenosine Deaminases: Advances and Challenges for Therapeutic RNA Editing.

Targeted transcriptome engineering, in contrast to genome engineering, offers a complementary and potentially tunable and reversible strategy for cellular engineering. In this regard, adenosine to inosine (A-to-I) RNA base editing was recently engineered to make programmable base conversions on target RNAs. Similar to the DNA base editing technology, A-to-I RNA editing may offer an attractive alternative in a therapeutic setting, especially for the correction of point mutations. This Perspective introduces five currently characterized RNA editing systems and serves as a reader's guide for implementing an appropriate RNA editing strategy for applications in research or therapeutics.

In vivo RNA editing of point mutations via RNA-guided adenosine deaminases.

We present in vivo sequence-specific RNA base editing via adenosine deaminases acting on RNA (ADAR) enzymes with associated ADAR guide RNAs (adRNAs). To achieve this, we systematically engineered adRNAs to harness ADARs, and comprehensively evaluated the specificity and activity of the toolsets in vitro and in vivo via two mouse models of human disease. We anticipate that this platform will enable tunable and reversible engineering of cellular RNAs for diverse applications.

Oligonucleotide conjugated multi-functional adeno-associated viruses.

Recombinant adeno-associated viruses (AAVs) are among the most commonly used vehicles for in vivo gene delivery. However, their tropism is limited, and additionally their efficacy can be negatively affected by prevalence of neutralizing antibodies in sera. Methodologies to systematically engineer AAV capsid properties would thus be of great relevance. In this regard, we develop here multi-functional AAVs by engineering precision tethering of oligonucleotides onto the AAV surface, and thereby enabling a spectrum of nucleic-acid programmable functionalities. Towards this, we engineered genetically encoded incorporation of unnatural amino acids (UAA) bearing bio-orthogonal chemical handles onto capsid proteins. Via these we enabled site-specific coupling of oligonucleotides onto the AAV capsid surface using facile click chemistry. The resulting oligo-AAVs could be sequence specifically labeled, and also patterned in 2D using DNA array substrates. Additionally, we utilized these oligo conjugations to engineer viral shielding by lipid-based cloaks that efficaciously protected the AAV particles from neutralizing serum. We confirmed these 'cloaked AAVs' retained full functionality via their ability to transduce a range of cell types, and also enable robust delivery of CRISPR-Cas9 effectors. Taken together, we anticipate this programmable oligo-AAV system will have broad utility in synthetic biology and AAV engineering applications.