RNAi-mediated rheostat for dynamic control of AAV-delivered transgenes

Abstract

Adeno-associated virus (AAV)-based gene therapy could be facilitated by the development of molecular switches to control the magnitude and timing of expression of therapeutic transgenes. RNA interference (RNAi)-based approaches hold unique potential as a clinically proven modality to pharmacologically regulate AAV gene dosage in a sequence-specific manner. We present a generalizable RNAi-based rheostat wherein hepatocyte-directed AAV transgene expression is silenced using the clinically validated modality of chemically modified small interfering RNA (siRNA) conjugates or vectorized co-expression of short hairpin RNA (shRNA). For transgene induction, we employ REVERSIR technology, a synthetic high-affinity oligonucleotide complementary to the siRNA or shRNA guide strand to reverse RNAi activity and rapidly recover transgene expression. For potential clinical development, we report potent and specific siRNA sequences that may allow selective regulation of transgenes while minimizing unintended off-target effects. Our results establish a conceptual framework for RNAi-based regulatory switches with potential for infrequent dosing in clinical settings to dynamically modulate expression of virally-delivered gene therapies.

Suppression of toxic transgene expression by optimized artificial miRNAs increases AAV vector yields in HEK-293 cells

“…Here, the expression of the transgene is induced by inhibiting the silencing shRNA via a high-affinity oligonucleotide complementary to the shRNA sequence. 39 …”

Brainwide silencing of prion protein by AAV-mediated delivery of an engineered compact epigenetic editor

“…AAV-mediated delivery of transgenes in nondividing cells results in chronic expression from episomal AAV genomes, raising antigenicity and off-target editing concerns. As a result, previous efforts have aimed to restrict AAV expression once the desired therapeutic edits are accomplished (74)(75)(76). However, in the case of CRISPR cutting, this is at the cost of increased AAV transgene genomic insertions.…”

AAV Immunotoxicity: Implications in Anti-HBV Gene Therapy

“…Transgene expression from weaker hepatocyte-specific promoters and RNAi-mediated transgene self-regulation [140][141][142] Avoid TLR 9 signaling activation CpG island reduction or TLR9 inhibition via oligonucleotide incorporation in the genome [143][144][145] Avoid dsRNA sensing Engineering of the expression cassette, such that transcription from 3 ITR is inhibited [146] Repress the activation of primary and memory T-cell responses Co-expressing NS5A with the transgene [147] Capsid Reduce dose, de-target APCs, and induce tolerance Rational designs and directed evolution to generate capsids with high hepatocyte transduction efficiencies [148][149][150][151] Avoid complement activation Rational designs and directed evolution to generate capsids that will evade pre-existing antibodies [135,[152][153][154] Avoid TLR9 signaling activation TLR9 inhibiting peptides insertion in the capsid [155] Reduce antigen presentation Mutation of surface-exposed tyrosine residues [156,157] Enhance Treg proliferation and reduce CD8+ T cell activity Incorporation of the MHC II epitopes derived from IgG in the AAV capsid [158] 11.…”

“…Recent studies have harnessed RNAi to design self-regulating AAVs and avoid transgene expression in specific cell types. Vectors can be made to carry a target of the tissuespecific miRNA or can be designed to co-express tissue-specific promoter-driven shRNA or pri-miRNA together with transgene mRNAs bearing the target site to the encoded shRNA/pri-miRNA [142]. This concept has been clearly demonstrated in CNS-targeting AAVs.…”