Future of rAAV Gene Therapy: Platform for RNAi, Gene Editing, and Beyond

Abstract

The use of recombinant adeno-associated viruses (rAAVs) ushered in a new millennium of gene transfer for therapeutic treatment of a number of conditions, including congenital blindness, hemophilia, and spinal muscular atrophy. rAAV vectors have remarkable staying power from a therapeutic standpoint, withstanding several ebbs and flows. As new technologies such as clustered regularly interspaced short palindromic repeat genome editing emerge, it is now the delivery tool-the AAV vector-that is the stalwart. The long-standing safety of this vector in a multitude of clinical settings makes rAAV a selling point in the advancement of approaches for gene replacement, gene knockdown, gene editing, and genome modification/engineering. The research community is building on these advances to develop more tailored delivery approaches and to tweak the genome in new and unique ways. Intertwining these approaches with newly engineered rAAV vectors is greatly expanding the available tools to manipulate gene expression with a therapeutic intent.

Keywords: AAV vectors; RNAi; genome editing; miRNA; non-coding RNA.

Figure 1.

Broad categories of recombinant adeno-associated virus therapeutic strategies. (A) Gene delivery, often of a cDNA that replaces a missing or defective gene, driven by an exogenous promoter. (B) Gene knockdown through delivery of sequences that generate small interfering RNAs that can degrade a mutant gene or infectious virus. (C) Genome editing by zinc finger nucleases, transcription activator-like effector nucleases, or the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system to introduce or correct mutations in the host or virus genome or introduce transcripts at double-stranded genome breaks. (D) Genome modulation through introduction of DNA sequences with homology to the host genome to correct a mutation or provide a therapeutic gene. (E) Non-coding RNA modulation or sequestration to alter transcript and protein levels, for example through introduction of sequences with engineered binding sites.

Figure 2.

Broad categories of CRISPR/Cas9 genome editing strategies. (A) The use of the CRISPR/Cas9 with a single guide RNA (sgRNA) targeting a specific genomic region introduces double-stranded breaks that lead to insertions and deletions (indels) through the non-homologous end joining pathway. A donor template can be co-delivered to introduce a specific DNA segment at the double-stranded break by homology directed repair. (B) Cas9 nickase enzymes have a mutation that directs single-stranded DNA breaks. When two sgRNAs are engineered to target regions in a reasonable proximity along with Cas9 nickase enzymes, off-targeting is reduced because the chance that similar off-targets are close by is low. This also facilitates larger genomic deletions. (C) The use of catalytically inactive Cas9 (dCas9) can leverage the recruitment of the enzyme tethered to an additional transcription factor to modulate the genome or epigenome without introducing breaks. In all situations, the use of patient-specific or common single nucleotide polymorphisms can direct editing to the mutant allele.