Use of HSC Targeted LNP to Generate a Mouse Model of Lethal α-Thalassemia and Treatment via Lentiviral Gene Therapy.

-Thalassemia (AT) is one of the most commonly occurring inherited hematological diseases. However, few treatments are available, and allogeneic bone marrow transplantation (BMT) is the only available therapeutic option for patients with severe AT. Research into AT has remained limited due to a lack of adult mouse models, with severe AT typically resulting in in utero lethality. By using a lipid nanoparticle (LNP) targeting the receptor CD117 and delivering a Cre mRNA (mRNACreLNPCD117), we were able to delete floxed -globin genes at high efficiency in hematopoietic stem cells (HSC) ex vivo. These cells were then engrafted in the absence or presence of a novel α-globin expressing lentiviral vector (ALS20I). Myeloablated mice transplanted with mRNACreLNPCD117-treated HSC showed a complete knockout of -globin genes. They demonstrated a phenotype characterized by the synthesis of hemoglobin H (-tetramers,  or HbH), aberrant erythropoiesis, and abnormal organ morphology, culminating in lethality approximately eight weeks following engraftment. Mice receiving mRNACreLNPCD117-treated HSC with at least one copy of ALS20I survived long-term with normalization of erythropoiesis, decreased the production of HbH, and ameliorated the abnormal organ morphology. Furthermore, we tested ALS20I in erythroid progenitors derived from -globin-KO CD34+ and cells isolated from patients with both deletional and non-deletional HbH disease, demonstrating improvement in -globin/-globin mRNA ratio and reduction in the formation of HbH by HPLC. Our results demonstrate the broad applicability of LNP for disease modeling, characterization of a novel severe mouse model of AT, and the efficacy of ALS20I for treating AT.

SARS-CoV-2 evolution during prolonged infection in immunocompromised patients.

Prolonged infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in immunocompromised patients provides an opportunity for viral evolution, potentially leading to the generation of new pathogenic variants. To investigate the pathways of viral evolution, we carried out a study on five patients experiencing prolonged SARS-CoV-2 infection (quantitative polymerase chain reaction-positive for 79-203 days) who were immunocompromised due to treatment for lymphoma or solid organ transplantation. For each timepoint analyzed, we generated at least two independent viral genome sequences to assess the heterogeneity and control for sequencing error. Four of the five patients likely had prolonged infection; the fifth apparently experienced a reinfection. The rates of accumulation of substitutions in the viral genome per day were higher in hospitalized patients with prolonged infection than those estimated for the community background. The spike coding region accumulated a significantly greater number of unique mutations than other viral coding regions, and the mutation density was higher. Two patients were treated with monoclonal antibodies (bebtelovimab and sotrovimab); by the next sampled timepoint, each virus population showed substitutions associated with monoclonal antibody resistance as the dominant forms (spike K444N and spike E340D). All patients received remdesivir, but remdesivir-resistant substitutions were not detected. These data thus help elucidate the trends of emergence, evolution, and selection of mutational variants within long-term infected immunocompromised individuals. IMPORTANCE: SARS-CoV-2 is responsible for a global pandemic, driven in part by the emergence of new viral variants. Where do these new variants come from? One model is that long-term viral persistence in infected individuals allows for viral evolution in response to host pressures, resulting in viruses more likely to replicate efficiently in humans. In this study, we characterize replication in several hospitalized and long-term infected individuals, documenting efficient pathways of viral evolution.

A comparison of multivalent and bivalent vaccination strategies for the control of virulent ovine footrot.

Virulent footrot is a significant economic and animal welfare concern. The disease can be treated, controlled, and eliminated with vaccine, but selecting the appropriate vaccination strategy can be challenging. There are two main strategies: outbreak (serogroup)-specific univalent or bivalent vaccination, or use of a multivalent vaccine containing up to nine of the most common serogroups. The objective of this study was to compare these approaches in sheep flocks infected with multiple Dichelobacter nodosus serogroups. In the first phase, we undertook an immunogenicity trial in which we compared four pre-commercial multivalent recombinant fimbrial vaccines containing six (A, B, C, G, H, I) or nine (A, B, C, D, E, F, G, H, I) serogroups, and compared them to commercial bivalent vaccines. Two multivalent vaccines stimulated significantly higher antibody responses than two other multivalent vaccines but the number of serogroups included in the multivalent vaccine formulations did not have a significant effect. In the first phase, we also compared inter-vaccination intervals of two- and three-months between sequential bivalent vaccines, and found that a two-month interval was sufficient to avoid antigenic competition. In the second phase, the most immunogenic multivalent vaccine (nine serogroups) was compared to sequential bivalent vaccines and monthly foot-bathing in a field trial in four commercial Merino flocks. The duration of protection afforded by the multivalent vaccine was likely to be less than that of the bivalent vaccines, as the antibody titres stimulated were lower and less persistent.