Some recombinant adeno-associated viruses (rAAVs) can cross the neonatal blood-brain barrier (BBB) and efficiently transduce cells of the central nervous system (CNS). However, in the adult CNS, transduction levels by systemically delivered rAAVs are significantly reduced, limiting their potential for CNS gene therapy. Here, we characterized 12 different rAAVEGFPs in the adult mouse CNS following intravenous delivery. We show that the capability of crossing the adult BBB and achieving widespread CNS transduction is a common character of AAV serotypes tested. Of note, rAAVrh.8 is the leading vector for robust global transduction of glial and neuronal cell types in regions of clinical importance such as cortex, caudate-putamen, hippocampus, corpus callosum, and substantia nigra. It also displays reduced peripheral tissue tropism compared to other leading vectors. Additionally, we evaluated rAAVrh.10 with and without microRNA (miRNA)-regulated expressional detargeting from peripheral tissues for systemic gene delivery to the CNS in marmosets. Our results indicate that rAAVrh.8, along with rh.10 and 9, hold the best promise for developing novel therapeutic strategies to treat neurological diseases in the adult patient population. Additionally, systemically delivered rAAVrh.10 can transduce the CNS efficiently, and its transgene expression can be limited in the periphery by endogenous miRNAs in adult marmosets.

No abstract

Recombinant adeno-associated viruses (rAAVs) are currently considered the safest and most reliable gene delivery vehicles for human gene therapy. Three serotype capsids, AAV1, AAV2, and AAV9, have been approved for commercial use in patients, but they may not be suitable for all therapeutic contexts. Here, we describe a novel capsid identified in a human clinical sample by high-throughput, long-read sequencing. The capsid, which we have named AAVv66, shares high sequence similarity with AAV2. We demonstrate that compared to AAV2, AAVv66 exhibits enhanced production yields, virion stability, and CNS transduction. Unique structural properties of AAVv66 visualized by cryo-EM at 2.5-Å resolution, suggest that critical residues at the threefold protrusion and at the interface of the five-fold axis of symmetry likely contribute to the beneficial characteristics of AAVv66. Our findings underscore the potential of AAVv66 as a gene therapy vector.

The central nervous system (CNS) is a fascinating and intricate set of biological structures that we have yet to fully understand. Studying the in vivo function of the CNS and finding novel methods for treating neurological disorders have been particularly challenging. One difficulty is correcting genetic disorders afflicting the CNS in a targeted manner. Recombinant adeno-associated viruses (rAAVs) have emerged as promising therapeutic tools for treating genetic defects of the CNS, due to their excellent safety profile and ability to cross the blood-brain barrier (BBB). While stereotactic injection of AAV is promising for localized gene delivery, it is less desirable for some applications because of the technique's invasiveness and limited intraparenchymal spread.1 Long-term expression of transgenes can be blocked by the host immune response. The transgene product can be recognized as nonself, triggering the activation of both humoral and cellmediated immune responses. This is both problematic for completely foreign gene products, as well as those that are mutated forms of endogenously expressed proteins. Transgene cassettes that carry miR-BS to recruit the antigen presenting cell (APC)-enriched miR-142, thereby blocking expression in dendritic cells, have shown great promise [50,51]. Such constructs are within the size limitations of rAAV and are compatible with ubiquitous or CNS-specific promoters that can achieve high-level expression. 3 There is strong evidence that many standard animal models harbor preexisting antibodies against AAV serotypes AAV1, AAV2, AAV6, and AAV9 [53]. Neutralizing antibodies (NAbs) against AAVs are present in the sera of mice, rats, rabbits, dogs, sheep, and pigs. The CNS has traditionally been considered an immune privileged organ. However, autoimmune diseases such as multiple sclerosis suggest that the CNS is not completely protected from inflammation [55]. Because targeting the CNS via IV injection will expose animals to vector on a systemic level, immune response should be heavily considered. Therefore, prescreening of animals for NAbs is recommended to assess vector effectiveness in all animal subjects, regardless of species. Of note, we and others have found that even NAb titers between 1:2 and 1:5 can prevent in vivo transduction by intravascular AAV9 [53]. Several studies have focused on developing novel capsids that can efficiently evade host immunity. High-throughput human IgG screening via directed evolution of capsids, in combination with the development of humanized liver mouse models, has identified capsids that can transduce human liver as well as exhibit low immunogenicity [56]. However, a capsid that can both bypass the BBB and evade the human host immune system has yet to be developed.