Abstract
Manipulating lymphocyte functions with gene silencing approaches is promising for treating autoimmunity, inflammation, and cancer. Although oligonucleotide therapy has been proven to be successful in treating several conditions, efficient in vivo delivery of oligonucleotide to lymphocyte populations remains a challenge. Here, we demonstrate that intravenous injection of a heteroduplex oligonucleotide (HDO), comprised of an antisense oligonucleotide (ASO) and its complementary RNA conjugated to α-tocopherol, silences lymphocyte endogenous gene expression with higher potency, efficacy, and longer retention time than ASOs. Importantly, reduction of Itga4 by HDO ameliorates symptoms in both adoptive transfer and active experimental autoimmune encephalomyelitis models. Our findings reveal the advantages of HDO with enhanced gene knockdown effect and different delivery mechanisms compared with ASO. Thus, regulation of lymphocyte functions by HDO is a potential therapeutic option for immune-mediated diseases.
Highlights
Manipulating lymphocyte functions with gene silencing approaches is promising for treating autoimmunity, inflammation, and cancer
Specific antisense oligonucleotide (ASO) sequences for Malat[1] and dystrophia myotonica-protein kinase (Dmpk) have high specificity, efficacy, biological stability, and safety in previous studies[19,20,21] (Supplementary Fig. 1b)
We evaluated the in vivo gene silencing efficacy of Toc-ASO, but all mouse died within 2 days after intravenous injection of Toc-ASO at corresponding doses to 50 mg/kg of the ASO, indicating high toxicity with direct conjugation of αtocopherol to ASO in vivo
Summary
Manipulating lymphocyte functions with gene silencing approaches is promising for treating autoimmunity, inflammation, and cancer. Most therapeutic ASO have phosphorothioate backbone modifications to enhance protein binding, stability in serum, and cellular uptake, presumably through ASO binding to serum or cell surface proteins[3] Despite these chemical modifications, physical or chemical transfection methods such as electroporation or nucleofection are required for transduction of ASO into lymphocytes[4,5], typically leading to significantly decreased cell viability because of transient membrane permeabilization[6,7,8]. Other gene silencing techniques commonly applied to lymphocytes include nanoparticle- and aptamer-based interfering RNA delivery systems[9,10,11] These studies support the development of targeted oligonucleotide delivery platforms for therapeutics of lymphocyte-based diseases. Given that ASOs are actively developed oligonucleotide agents[17], our HDO technology equipped with α-tocopherol has potential for gene silencing in lymphocytes in vivo. This study provides a productive modality for administrating a chemically synthesized oligonucleotide that can silence target genes in mouse lymphocytes with high efficacy and safety
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