Enhanced selective gene delivery to neural stem cells in vivo by an adeno-associated viral variant

Abstract

Neural stem cells (NSCs) are defined by their ability to self-renew and to differentiate into mature neuronal and glial cell types. NSCs are the subject of intense investigation, owing to their crucial roles in neural development and adult brain function and because they present potential targets for gene and cell replacement therapies following injury or disease. Approaches to specifically genetically perturb or modulate NSC function would be valuable for either motivation. Unfortunately, most gene delivery vectors are incapable of efficient or specific gene delivery to NSCs in vivo. Vectors based on adenoassociated virus (AAV) present a number of advantages and have proven increasingly successful in clinical trials. However, natural AAV variants are inefficient in transducing NSCs. We previously engineered a novel AAV variant (AAV r3.45) capable of efficient transduction of adult NSCs in vitro. Here, to build upon the initial promise of this variant, we investigated its in vitro and in vivo infectivity. AAV r3.45 was more selective for NSCs than mature neurons in a human embryonic stem cell-derived culture containing a mixture of cell types, including NSCs and neurons. It was capable of more efficient and selective transduction of rat and mouse NSCs in vivo than natural AAV serotypes following intracranial vector administration. Delivery of constitutively active β-catenin yielded insights into mechanisms by which this key regulator modulates NSC function, indicating that this engineered AAV variant can be harnessed for preferential modulation of adult NSCs in the hippocampus. The capacity to rapidly genetically modify these cells might greatly accelerate in vivo investigations of adult neurogenesis.

Engineered AAV vectors for improved central nervous system gene delivery

“…Approximately 65% of the cells transduced in the rat hippocampus by AAV r3.45 were Type 2a NSCs, and 9% were Type 1 NSCs. 18 Furthermore, 60% of Type 2a NSCs and 41% of the Type 1 NSCs, respectively, were transduced by AAV r3.45 in the rat brain. 18 Similarly, in the mouse brain approximately 38% of Type 2a NSCs were transduced in the hippocampus.…”

“… 18 Furthermore, 60% of Type 2a NSCs and 41% of the Type 1 NSCs, respectively, were transduced by AAV r3.45 in the rat brain. 18 Similarly, in the mouse brain approximately 38% of Type 2a NSCs were transduced in the hippocampus. 18 …”

“… 17 A subsequent study showed that within an hESC-derived culture containing a mixture of cells including NSCs, neurons, and astrocytes, the percentage of NSCs infected by AAV r3.45 was significantly higher compared to wild-type AAV. 18 The variants still bound heparan sulfate proteoglycans (HSPG, AAV2s primary receptor), though peptide insertion at amino acid 588 in the parental AAV2 capsid lowered AAV 3.45s HSPG affinity. In addition, the inserted peptide, which did not have homology to any known cell surface proteins, likely conferred affinity to an undetermined secondary receptor that potentially mediated viral internalization.…”

“… 18 Similarly, in the mouse brain approximately 38% of Type 2a NSCs were transduced in the hippocampus. 18 …”

Combining Engineered Nucleases with Adeno-associated Viral Vectors for Therapeutic Gene Editing

“…Specifically, viral delivery of a HDR construct with homology to a chromosomal locus, even without a nuclease component, can result in recombination into the target locus at a rate of up to 1% [32], a rate >1,000-fold higher than conventional plasmid donors [33] or other viral vectors [34]. Successful AAV-mediated gene targeting has been achieved in neural stem cells [35], human pluripotent stem cells [36], and hematopoietic stem cells (HSCs) [37], among other cell types. In addition, as discussed below, combining AAV with a nuclease offers even stronger potential.…”