Fabb, S. | bioRxiv Science

bioRxiv2024

A Versatile Antibody Capture System that Drives Precise In Vivo Delivery of mRNA loaded Lipid Nanoparticles and Enhances Gene Expression

Efficient and precise delivery of mRNA is critical to advance mRNA therapies beyond their current use as vaccines. Lipid nanoparticles (LNP) efficiently encapsulate and protect mRNA, but non-specific cellular uptake may lead to off-target delivery and minimal delivery to target cells. Functionalizing LNPs with antibodies enables targeted mRNA delivery, but traditional modification techniques require complex conjugation and purification, which often reduces antibody affinity. Here, we present a simple method for capturing antibodies in their optimal orientation on LNPs, without antibody modification or complex purification. This strategy uses an optimally oriented anti-Fc nanobody on the LNP surface to capture antibodies, resulting in protein expression levels >1000 times higher than non-targeted LNPs and >8 times higher than conventional antibody functionalization techniques. These precisely targeted LNPs showed highly efficient in vivo targeting to T cells, with minimal delivery to other immune cells. This approach enables the rapid development of targeted LNPs and has the potential to broaden the use of mRNA therapies.

bioengineering↗

bioRxiv2023

Dysregulated expression of Hoxa1 isoforms in hematopoietic stem and progenitor cells causes myelodysplastic syndromes.

The homeobox gene, Hoxa1, has two different isoforms generated by alternative splicing: a full-length homeodomain-containing Hoxa1 (Hoxa1-FL), and a truncated Hoxa1 (Hoxa1-T), that lacks the homeodomain. The effects of the distinct Hoxa1 isoforms in hematopoiesis have not been investigated. Oncoretroviral studies revealed that Hoxa1-T acts in a dominant negative manner, regulating transcriptionally active Hoxa1. Oncoretroviral overexpression of wildtype Hoxa1 (WT-Hoxa1), which generates both Hoxa1 isoforms, in murine hematopoietic stem and progenitor cells (HSPCs) perturbed hematopoiesis, resulting in transplantable myelodysplastic syndromes (MDS) in mice. Overexpression of a mutated Hoxa1 cDNA (MUT-Hoxa1) that generates Hoxa1-FL, but not Hoxa1-T, led to a more severe MDS that transformed to secondary acute myeloid leukemia (sAML). DNA damage repair pathways were downregulated in Hoxa1-overexpressing hematopoietic progenitor cells, accompanied by increased {gamma}H2AX foci. In silico analyses revealed that CD34+ cells from approximately 50% of patients with MDS had elevated HOXA1-FL expression. Conditional knock-in WT-Hoxa1 and MUT-Hoxa1 mice were generated and had features of pre-MDS, developing altered hematopoiesis within 4 months of Hoxa1 isoform overexpression in HSPCs. HSPCs were significantly reduced in all knock-in mice, accompanied by significantly increased apoptosis in WT-Hoxa1 HSPCs. Healthy wildtype recipients transplanted with bone marrow cells from Hoxa1 knock-in mice developed trilineage MDS, with Hoxa1 isoform and gene dosage dependent phenotypes. Collectively our data identify a role for HOXA1 in the pathogenesis of MDS. Our Hoxa1 mouse models capture different stages of progression of disease from pre-MDS to MDS to sAML and provide novel, clinically relevant tools to study MDS. Key pointsHOXA1 is upregulated in approximately 50% of MDS patient CD34+ BM cells, highlighting a potential role for HOXA1 in the pathogenesis of MDS. Dysregulated expression of Hoxa1 isoforms in murine hematopoietic stem and progenitor cells predisposes mice to pre-MDS and MDS.

cancer biology↗