Biology subjects

Tanowitz, M.

Publications and source records attributed to Tanowitz, M..

3 recordsLinked to original sources

bioRxiv2026

Delivery of small interfering RNA and antisense oligonucleotides across the blood-brain barrier with monovalent transferrin receptor 1 binding VHH-Fc fusion proteins

The blood-brain barrier (BBB) is a highly selective cell layer that restricts the diffusion of diverse chemical entities into the central nervous system (CNS) from systemic circulation. Macromolecular therapeutics including oligonucleotides, peptides, and monoclonal antibodies exhibit only minimal brain distribution after systemic dosing due to exclusion by the BBB. Receptor-mediated transcytosis (RMT) has evolved to transport vital cargo across the BBB through a specialized vesicular transport pathway. Transferrin receptor 1 (TfR1) shuttles transferrin, its natural ligand, across the BBB, as well as TfR1-binding IgG antibodies and conjugates. Here, we describe a novel monovalent TfR1-binding VHH-Fc for the delivery of oligonucleotide cargo, including antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) across the BBB in rodents and non-human primates (NHPs), supporting the translational potential of the VHH-antisense RMT platform for the treatment of neurological disorders. We explore the role of binding affinity, conjugation site, drug-antibody ratio (DAR), and conjugation chemistry, and determine that binding affinity, DAR and conjugation site are major determinants of RMT capacity and brain activity of siRNAs delivered across the BBB. Graphical Abstract / Highlights O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/744307v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@d1d648org.highwire.dtl.DTLVardef@4b22d3org.highwire.dtl.DTLVardef@db8b6borg.highwire.dtl.DTLVardef@19e5ac3_HPS_FORMAT_FIGEXP M_FIG C_FIG - Anti-TfR1 (-TfR1) VHH ligands formatted as heterodimeric, 2-chain monovalent VHH-Fc were engineered for conjugation to siRNA and ASO. - Systematic in vivo evaluation of VHH clones spanning a range of TfR1 binding affinities revealed a relationship between TfR1 binding affinity and the CNS activity of intravenously dosed VHH-Fc-siRNA conjugates. - By optimizing TfR1 binding affinity, conjugation site, and conjugation chemistry, we identified VHH-Fc-siRNA molecules that efficiently cross the BBB via receptor-mediated transcytosis and reduce target mRNA across CNS tissues, including deeper brain regions, after intravenous (IV) or subcutaneous (SC) dosing in mice and non-human primates (NHPs).

neuroscience↗

bioRxiv2026

Systemic and Local Delivery of siRNA to the CNS and Periphery via Anti-IGF1R Antibody Conjugation

siRNA delivery platforms capable of accessing both central and peripheral tissues are critically needed to expand the therapeutic potential of oligonucleotides. To address this, we developed a novel siRNA-antibody conjugate by attaching an Hprt-targeting siRNA to an engineered antibody shuttle. This shuttle targets the insulin-like growth factor 1 receptor (IGF1R) using a fused antibody fragment (Clone F) and utilizes an antibody backbone with no tissue-relevant binding in this study. The resulting conjugate, designated Clone F-Hprt, demonstrated robust in vivo knockdown across multiple tissues. Clone F-Hprt demonstrated enhanced penetration into central nervous system (CNS) tissues compared to unconjugated siRNA following intracerebroventricular (ICV) and intravenous (IV) administration. In peripheral tissues, Clone F-Hprt achieved widespread knockdown in muscle, heart, and lung, consistent with IGF1R expression. The conjugate was well tolerated across all routes, including with repeated dosing. Although several receptor-mediated approaches for CNS delivery are progressing to the clinic (e.g., targeting the transferrin receptor), clinical validation remains to be demonstrated. Our findings highlight IGF1R as an alternative receptor capable of supporting delivery across both central and peripheral tissues, offering a complementary strategy for expanding the therapeutic landscape of oligonucleotide delivery.

neuroscience↗

bioRxiv2026

Tuning siRNA Specificity through Seed Region Incorporation of Deoxyribonucleotide Stereoisomers

Precise chemical design continues to drive advances in RNA-based therapeutics. Here, we report the synthesis and site-specific incorporation of four canonical phosphoramidites (U, C, A, and G), each bearing non-natural nucleoside configurations: {beta}-D-2'-deoxyxylonucleosides and -L-2'-deoxyribonucleosides. These stereochemically distinct nucleoside analogs were introduced at positions 6 and 7 within siRNA seed regions. When applied to siRNAs targeting Ttr, ACTN1, and Marc1, these modifications reduced off-target gene repression in functional assays and, in several cases, in transcriptome-wide differential expression analyses, while preserving robust on-target activity. In vivo, Marc1-targeting siRNAs containing these modified nucleosides showed decreased hepatotoxicity, as evidenced by reduced serum ALT and AST levels. Collectively, these findings establish {beta}-D-2'-deoxyxylonucleoside and -L-2'-deoxyribonucleoside analogs as promising chemical tools for enhancing the specificity and safety of siRNA therapeutics. This work underscores the power of integrating rational nucleoside design with comprehensive functional and in vivo evaluation to advance drug development based on RNA interference (RNAi). GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/699368v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@10e6e8aorg.highwire.dtl.DTLVardef@7b2797org.highwire.dtl.DTLVardef@1643f44org.highwire.dtl.DTLVardef@75a0a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗