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Genome editing driven by zinc-finger nucleases (ZFNs) yields high gene-modification efficiencies (>10%) by introducing a recombinogenic double-strand break into the targeted gene. The cleavage event is induced using two custom-designed ZFNs that heterodimerize upon binding DNA to form a catalytically active nuclease complex. Using the current ZFN architecture, however, cleavage-competent homodimers may also form that can limit safety or efficacy via off-target cleavage. Here we develop an improved ZFN architecture that eliminates this problem. Using structure-based design, we engineer two variant ZFNs that efficiently cleave DNA only when paired as a heterodimer. These ZFNs modify a native endogenous locus as efficiently as the parental architecture, but with a >40-fold reduction in homodimer function and much lower levels of genome-wide cleavage. This architecture provides a general means for improving the specificity of ZFNs as gene modification reagents.

Messenger ribonucleic acid (mRNA)‐based therapies, including conventional linear mRNA (linRNA), circular RNA (circRNA), and self‐amplifying RNA (saRNA), are being developed not only for vaccination but also for protein replacement, gene editing, and regenerative medicine. However, these mRNA modalities differ in structure and function, and their interactions with current non‐viral delivery systems influence their therapeutic efficacy. Here, the in vivo expression kinetics of linRNA, circRNA, and saRNA delivered via lipid nanoparticles (LNPs) or bioreducible poly(cystamine bisacrylamide‐co‐4‐amino‐1‐butanol) (pABOL) polymer are systematically evaluated. At 0.5 µg, Venezuelan equine encephalitis virus (VEEV)‐based saRNA resulted in higher total luciferase expression than 5 µg of linRNA or circRNA highlighting its superior potency. LNPs significantly enhanced expression of non‐amplifying mRNAs compared to pABOL, whereas pABOL delivery of saRNA yielded a ∼2‐fold improvement over LNPs. Furthermore, saRNAs derived from New World alphaviruses expressed 2–6 times more protein than Old World saRNAs when delivered with LNPs; these differences are not observed with pABOL. These findings demonstrate that mRNA modality, saRNA genotype, and delivery platform interact to determine therapeutic protein output. This study provides actionable insights for optimizing mRNA‐based therapeutics across diverse clinical applications. Self‐amplifying (saRNA), linear (linRNA), and circular (circRNA) mRNAs are compared under standardized conditions using lipid nanoparticles (LNPs) and pABOL polymer. saRNA achieved superior expression, while linRNA and circRNA performance varied based on untranslated region elements and delivery method. Differences between alphavirus saRNAs are enhanced by LNPs but attenuated with pABOL. These findings support rational selection of RNA modality–delivery platform combinations.

Background The benefits of mRNA-based platforms, such as rapid response and simplified manufacturing, may be overshadowed by lack of durable protective immunity compared to traditional vaccine technologies targeting certain pathogens. Self-replicating RNA has the potential to induce durable immune responses at lower doses than traditional mRNA. A recent Phase 1 clinical trial showed that a self-replicating RNA vaccine encoding rabies, RBI-4000, was able to show de novo immunogenicity at all doses tested, specifically 0.1, 1, and 10 micrograms in a prime-boost regimen or a single 10 microgram dose (NCT06048770). Methods Here, we report the secondary outcome of the Phase 1 study, durability of immune responses elicited by RBI-4000, as assessed by the presence of the rabies virus neutralizing antibody response, up to 8 months post immunization. We compare long term immunogenicity of RBI-4000 to a commercial comparator, an inactivated viral vaccine RabAvert, using several statistical models with a post-hoc analysis. The trial was performed at two sites in the United States enrolling 89 healthy volunteers aged 18-45. Results Individual rabies virus neutralizing antibody titers, above the benchmark seropositivity, were detected out to 8 months in all study cohorts. Statistical decay modeling showed that RBI-4000 induces rabies virus neutralizing antibodies with similar or improved durability compared to RabAvert. Conclusions We report the first durability data from a head-to-head study of an optimized self-replicating RNA vaccine for rabies that elicits sustained immune responses compared to a commercial comparator that uses a traditional vaccine technology. Plain language summary New vaccine types, like mRNA vaccines, can be made quickly and are helpful during outbreaks. But sometimes, they don’t give long-lasting protection, as seen with some COVID-19, flu, and common cold vaccines. We studied a new kind of vaccine called a self-replicating RNA (srRNA) vaccine, which may help the body build stronger and longer-lasting immunity. We tested an srRNA rabies vaccine, called RBI-4000, and compared it to a commonly used rabies vaccine called RabAvert. Over 8 months, we find that RBI-4000 worked as well or even better than RabAvert at keeping the immune response strong. This suggests that srRNA vaccines could be a useful new way to protect people from diseases for longer periods of time. Maine et al., report post-hoc modeling of a secondary outcome of a Phase 1 clinical trial that assessed the long-term immunogenicity of a next-generation self-replicating RNA vaccine, RBI-4000 for rabies. The durability of immune responses from RBI-4000 is equal to or superior to a commercial vaccine using statistical decay modeling.

RNA technology has recently come to the forefront of innovative medicines and is being explored for a wide range of therapies, including prophylactic and therapeutic vaccines, biotherapeutic protein expression and gene therapy. In addition to conventional mRNA platforms now approved for prophylactic SARS-CoV2 vaccines, synthetic self-replicating RNA vaccines are currently being evaluated in the clinic for infectious disease and oncology. The prototypical srRNA vectors in clinical development are derived from alphaviruses, specifically Venezuelan Equine Encephalitis Virus (VEEV). While non-VEEV alphaviral strains have been explored as single cycle viral particles, their use as synthetic vectors largely remains under-utilized in clinical applications. Here we describe the potential commonalities and differences in synthetic alphaviral srRNA vectors in host cell interactions, immunogenicity, cellular delivery, and cargo expression. Thus, unlike the current thinking that VEEV-based srRNA is a one-size-fits-all platform, we argue that a new drug development approach leveraging panels of customizable, synthetic srRNA vectors will be required for clinical success.

Activated B cells can tailor their ensuing antibody responses by isotype switching. McHeyzer-Williams and colleagues demonstrate B cell–intrinsic requirements for the transcription factors T-bet and RORα in maintaining IgG2a + and IgA + memory cells, respectively. Antibody class defines function in B cell immunity, but how class is propagated into B cell memory remains poorly understood. Here we demonstrate that memory B cell subsets unexpectedly diverged across antibody class through differences in the effects of major transcriptional regulators. Conditional genetic deletion of the gene encoding the transcription factor T-bet selectively blocked the formation and antigen-specific response of memory B cells expressing immunoglobulin G2a (IgG2a) in vivo . Cell-intrinsic expression of T-bet regulated expression of the transcription factor STAT1, steady-state cell survival and transcription of IgG2a-containing B cell antigen receptors (BCRs). In contrast, the transcription factor RORα and not T-bet was expressed in IgA + memory B cells, with evidence that knockdown of RORα mRNA expression and chemical inhibition of transcriptional activity also resulted in lower survival and BCR expression of IgA + memory B cells. Thus, divergent transcriptional regulators dynamically maintain subset integrity to promote specialized immune function in class-specific memory B cells.

Self-replicating RNA (srRNA) technology, in comparison to mRNA vaccines, has shown dose-sparing by approximately 10-fold and more durable immune responses. However, no improvements are observed in the adverse events profile. Here, we develop an srRNA vaccine platform with optimized non-coding regions and demonstrate immunogenicity and safety in preclinical and clinical development. Optimized srRNA vaccines generate protective immunity (according to the WHO defined thresholds) at doses up to 1,000,000-fold lower than mRNA in female mouse models of influenza and rabies. Clinically, safety and immunogenicity of RBI-4000, an srRNA vector encoding the rabies glycoprotein, was evaluated in a Phase I study (NCT06048770). RBI-4000 was able to elicit de novo protective immunity in the majority of healthy participants when administered at a dose of 0.1, 1, or 10 microgram (71%, 94%, 100%, respectively) in a prime-boost schedule. Similarly, we observe immunity above the WHO benchmark of protection following a single administration in most participants at both 1 and 10 microgram doses. There are no serious adverse events reported across all cohorts. These data establish the high therapeutic index of optimized srRNA vectors, demonstrating feasibility of both low dose and single dose approaches for vaccine applications. Here the authors report an optimized self-replicating RNA (srRNA) vaccine approach that generates protective immunity at much lower doses than mRNA vaccines in mice. In a Phase 1 study using rabies glycoprotein as antigen, they show robust immune responses at doses as low as 0.1 µg, with a favorable safety profile.

Antibody class defines function in B cell immunity, but how class is propagated into B cell memory remains poorly understood. Here we demonstrate that memory B cell subsets unexpectedly diverged across antibody class through differences in the effects of major transcriptional regulators. Conditional genetic deletion of the gene encoding the transcription factor T-bet selectively blocked the formation and antigen-specific response of memory B cells expressing immunoglobulin G2a (IgG2a) in vivo. Cell-intrinsic expression of T-bet regulated expression of the transcription factor STAT1, steady-state cell survival and transcription of IgG2a-containing B cell antigen receptors (BCRs). In contrast, the transcription factor RORα and not T-bet was expressed in IgA(+) memory B cells, with evidence that knockdown of RORα mRNA expression and chemical inhibition of transcriptional activity also resulted in lower survival and BCR expression of IgA(+) memory B cells. Thus, divergent transcriptional regulators dynamically maintain subset integrity to promote specialized immune function in class-specific memory B cells.

Antibody class defines function in B cell immunity, but how class is propagated into B cell memory remains poorly understood. Here, we demonstrate that memory B cell subsets unexpectedly diverge across antibody class through the differential impact of major transcriptional regulators. Conditional genetic deletion of Tbx21 selectively blocks the formation and antigen-specific response of IgG2a memory B cells in vivo. Cell intrinsic T-bet expression regulates STAT1 expression, steady-state cell survival and IgG2a BCR transcription. In contrast, RORα was differentially expressed in IgA memory B cells with siRNA knockdown and chemical inhibition supporting its selective control in cell survival and IgA BCR transcription. Thus, divergent transcriptional regulators dynamically maintain subset integrity to promote specialized immune function within class-specific memory B cells.

Zinc-finger nucleases (ZFNs) drive highly efficient genome editing by generating a site-specific DNA double-strand break (DSB) at a predetermined site in the genome. Subsequent repair of this break via the non-homologous end-joining (NHEJ) or homology-directed repair (HDR) pathways results in targeted gene disruption or gene addition, respectively. Here we report that ZFNs can be engineered to induce a site-specific DNA single-strand break (SSB) or nick. Using the CCR5-specific ZFNs as a model system we show that introduction of a nick at this target site stimulates gene addition using a homologous donor template, but fails to induce significant levels of the small insertions and deletions (indels) characteristic of repair via NHEJ. Gene addition by these CCR5-targeted zinc finger nickases (ZFNickases) occurs in both transformed and primary human cells at efficiencies of up to ~1-8%. Interestingly, ZFNickases targeting the AAVS1 \"safe harbor\" locus reveal similar in vitro nicking activity, a marked reduction of indels characteristic of NHEJ but stimulated far lower levels of gene addition - suggesting that other, yet to be identified, mediators of nick induced gene targeting exist. Introduction of a site-specific nicks at distinct endogenous loci provide an important tool for the study of DNA repair. Moreover, the potential for a SSB to direct repair pathway choice (i.e. HDR but not NHEJ) may prove advantageous for certain therapeutic applications such as the targeted correction of human disease-causing mutations.

Memory B cells are long-lived antigen-experienced B cells that typically express a high-affinity B cell receptor and rapidly expand their populations and differentiate into plasma cells after antigen rechallenge. While classes of membrane-bound antibody have differing abilities to transduce signals through their BCR based on their constant region, little is known about the molecular signals required for memory B cell survival, activation, or differentiation. Furthermore, it is unknown whether there exist class-specific differences in transcriptional programming. Several studies have delineated how cytokines differentially affect transcriptional programs differently in naïve B cells that culminate in an irreversible genetic recombination; however, it remains to be studied whether programmatic differences initiated at the time of class switching extend into the memory B cell compartments to control longevity, cell fate, and memory B cell function in a class-specific manner. Here we focus on IgG2a+ and IgA+ memory B cells and provide evidence for the divergent programming of memory B cell function by the major transcriptional regulators T-bet and RORα. Temporal deletion of T-bet in IgG2a+ memory B cells established a central and selective role for this regulator in the survival and antigen responsiveness of IgG2a+ memory B cells in vivo. Differences in the expression of cytokine receptors, integrins and RORα highlighted the specialized development and unique properties of IgA+ memory B cells. Notably, a role for both T-bet and RORα in persistent BCR transcription indicated both dynamic and ongoing class-specific requirements for each of these factors in IgG2a+ and IgA+ memory B cells, respectively. Thus, we propose that expression of any non-IgM antibody signifies a transcriptionally regulated commitment event across subspecialized, class-specific memory B cells. This commitment event establishes unique, class-specific molecular programs and organizes memory B cells into discrete lineages with separate functions.