In Vivo Selection of a Computationally Designed SCHEMA AAV Library Yields a Novel Variant for Infection of Adult Neural Stem Cells in the SVZ

Ojala, David S.; Sun, Sabrina; Santiago-Ortiz, Jorge L.; Shapiro, Mikhail G.; Romero, Philip A.; Schaffer, David V.

doi:10.1016/j.ymthe.2017.09.006

Cited by 81 publications

(110 citation statements)

References 100 publications

(128 reference statements)

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“…Beyond neurons, ICV injections also provide access to periventricular cell populations. For example, after ICV injection, AAV4 can be used to transduce the ependymal cells (Liu et al, 2005), and two engineered AAV capsids, SCH9 and AAV4.18, enable transduction of subventricular zone neural progenitors (Murlidharan et al, 2015;Ojala et al, 2018). IT injection can be used to deliver genes to spinal cord motor neurons and dorsal root ganglions (Federici et al, 2012;Foust et al, 2013;Schuster et al, 2014). Retrograde and anterograde transport for circuit studies AAV vectors are also commonly used as part of circuit studies.…”

Section: Csf Deliverymentioning

confidence: 99%

“…Choosing the optimal serotype requires reviewing the literature most relevant to the planned experiment and performing pilot testing for new or at least for challenging applications. As new engineered capsids with unique features continue to be developed, the available options will become more numerous and more powerful (Chan et al, 2017;Davidsson et al, n.d.;Deverman et al, 2016;Ojala et al, 2018;Tervo et al, 2016)…”

Section: Csf Deliverymentioning

confidence: 99%

“…While powerful technologies are being developed, each has technical limits which need to be considered both when designing experiments and when interpreting results. To this end, improved platforms for sharing (Borel et al, 2016;Federici et al, 2012;Gurda et al, 2016;Samaranch et al, 2013) AAV4 enables transduction of ependymal cells (Liu et al, 2005) AAV SCH9 and AAV4.18 enable SVZ progenitor cell transduction (Murlidharan et al, 2015;Ojala et al, 2018) ( Gray et al, 2011;Klein et al, 1998;McCown et al, 1996;Paterna et al, 2000;Tenenbaum et al, 2004;Thomsen et al, 1984;Yaguchi et al, 2013) CAG, CMV enhancer, CBA promoter, globin intron Ubiquitous 1700 (Miyazaki et al, 1989) CAGGS, CMV immediateearly enhancer, CBA promoter, CBA intron1/exon1…”

Section: Areas For Developmentmentioning

confidence: 99%

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Adeno-Associated Virus Technologies and Methods for Targeted Neuronal Manipulation

Haery

Deverman

Matho

et al. 2019

Preprint

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Cell-type-specific expression of molecular tools and sensors is critical to construct circuit diagrams and to investigate the activity and function of neurons within the nervous system. Strategies for targeted manipulation include combinations of classical genetic tools such as Cre/loxP and Flp/FRT, use of cis-regulatory elements, targeted knock-in transgenic mice, and gene delivery by AAV and other viral vectors. The combination of these complex technologies with the goal of precise neuronal targeting is a challenge in the lab. This report will discuss the theoretical and practical aspects of combining current technologies and establish best practices for achieving targeted manipulation of specific cell types. Novel applications and tools, as well as areas for development, will be envisioned and discussed.Selecting the Route of Administration and Capsid AAV tropism, as dictated by AAV capsid proteins, is an important factor affecting transduction efficiency and specificity across cell types. Since the mechanism of AAV transduction is through interaction of the AAV capsid with cell surface proteins and glycans, protein composition of the capsid (i.e., the AAV serotype) and the cell surface (i.e., based on cell type) determines transduction efficiency. Consequently, serotype and route of delivery should be carefully considered when designing experiments (Fig 1A, B). For an overview of the primary receptors for AAV serotypes, see (Schultz and Chamberlain, 2008). Direct intraparenchymal deliveryWhen injected directly into the brain, many of the naturally-occurring AAV capsids, which share homology ranging from 65-99% (Drouin and Agbandje-McKenna, 2013), have distinct but significantly overlapping tropisms and distribution characteristics. AAV1, AAV2, AAV5, AAV8,

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Section: Csf Deliverymentioning

confidence: 99%

Section: Csf Deliverymentioning

confidence: 99%

Section: Areas For Developmentmentioning

confidence: 99%

See 1 more Smart Citation

Adeno-Associated Virus Technologies and Methods for Targeted Neuronal Manipulation

Haery

Deverman

Matho

et al. 2019

Preprint

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“…evolution has been used to generate novel capsids with altered tropism and function [1][2][3][4][5][6][7][8][9] . This approach, however, involves a selection process that requires multiple generations of screenings to identify real functional capsids [2][3][4] . Due to the random nature of this process, it is also inherently unreproducible, and the resulting capsid variants provide little mechanistic insights into the molecular targets engaged.…”

Section: Engineering Of Adeno-associated Viral (Aav) Vector Capsids Tmentioning

confidence: 99%

“…Capsid variant All viral preps were produced to package the same scAAV-CMV-GFP genome. Cells were transduced with an MOI of 1x10 4 and analyzed 72hours post transduction using fluorescence microscopy. Of note is that MNM001 was selected using the BRAVE approach through a screening in HEK293T cells and that MNM008 was broadly observed to have a high affinity for human cells both in vitro ( Supplementary Fig.…”

Section: Average Gfp Intensity [Au]mentioning

confidence: 99%

Barcoded Rational AAV Vector Evolution enables systematicin vivomapping of peptide binding motifs

Davidsson

Wang

Aldrin-Kirk

et al. 2018

Preprint

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Here, we have developed a novel high-throughput approach to engineering of AAV capsids that encompasses all the benefits of rational design [12][13][14][15][16] while maintaining the broad screening diversity permitted by the directed evolution. This is achieved using only a single screening step. In the first application of BRAVE screening, we utilize peptides derived from proteins with documented synapse interaction with the aim to develop novel AAV capsid variants that are efficiently taken up and transported retrogradely in neurons in vivo.This combinatorial method enabled the generation and simultaneous functional mapping of close to 4 million unique AAV virion variants in parallel in vitro and in vivo. In a single round of screening, we selected 25 candidates, all of which could be derived into functional viruses with the capacity to become retrogradely transported in vivo or efficiently transduce of neurons in vitro. We identified one novel capsid variant expressing highly efficient retrograde transport in both rat and human dopamine (DA) neurons in vivo, and a panneuronal retrogradely transported AAV which we utilized to elucidate the function of basolateral amygdala projections to the dorsal striatum.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/335372 doi: bioRxiv preprint first posted online May. 31, 2018; Davidsson et al., 2018 | In vivo single-generation AAV-screen 3 To link capsid structure to an in vivo expressed molecular barcode, we generated a novel AAV production plasmid. A barcode is inserted in the 3' UTR of GFP of a gutted selfcomplementary AAV genome 17, 18 and the AAV2 Rep/Cap genes are expressed from the same plasmid, creating a linkage between capsid structure and barcode. Here, peptides are displayed at N587 of VP1 capsid protein 13 ( Supplementary Fig. S1). This mutates the wildtype AAV2 heparan sulfate proteoglycan binding motif 14. The AAV virus produced from this plasmid with no inserted peptide is hereafter referred to as MNMnull.With the aim to targeted neurons at their terminals, we selected 131 proteins based on their documented affinity to synapses ( Fig. 1A and Supplementary Fig. S2 and S3). Their amino acid (aa) sequences were computationally digested into overlapping 14aa or 22aa long polypeptides and three alternative linkers were then added to the 14 aa polypeptides (1-2.in Fig. 1B). The resulting 92 358 oligonucleotides were synthesized in parallel on an array (3. in Fig. 1B). They were then assembled into the backbone plasmid to allow for packaging of replication deficient AAV viral particles, where the peptide is displayed on the capsid surface and a 20bp random molecular barcode (BC) is included as part of the genome (4.in Fig. 1B and Supplementary Fig. S1). In parallel, the same plasmid library was utilized to generate a Look-Up table (LUT), linking the random barc...

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AAV Production Everywhere: A Simple, Fast, and Reliable Protocol for In‐house AAV Vector Production Based on Chloroform Extraction

et al. 2020

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Recombinant adeno‐associated virus (rAAV) is a mammalian virus that has been altered to be used as a gene delivery vehicle. Several changes to the viral genome have made them replication deficient so that this aspect of the viral infection cycle is under full control of the experimenter, while maintaining gene expression machinery. Over the last decades, rAAVs have become the gold standard for studying in vivo gene function and are especially favorable for gene transfer in the central nervous system. AAVs have been proven safe and provide stable gene expression over a long period of time. They are extensively used in preclinical experiments and show great potential for clinical applications. However, the use of AAVs in preclinical settings are often held back due to availability. Waiting lines are long at commercial production facilities, and in‐lab production is hindered due to lack of specific laboratory equipment needed. Here we present a novel production method that can be carried out in any molecular biology laboratory using standard laboratory equipment. We provide a simple, fast, and streamlined protocol for production that can result in titers comparable with the more time‐consuming iodixanol gradient ultracentrifugation method. The yield using this protocol is high enough for any type of study where AAV is the vector of choice. © 2020 The Authors.

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In Vivo Selection of a Computationally Designed SCHEMA AAV Library Yields a Novel Variant for Infection of Adult Neural Stem Cells in the SVZ

Cited by 81 publications

References 100 publications

Adeno-Associated Virus Technologies and Methods for Targeted Neuronal Manipulation

Adeno-Associated Virus Technologies and Methods for Targeted Neuronal Manipulation

Barcoded Rational AAV Vector Evolution enables systematicin vivomapping of peptide binding motifs

AAV Production Everywhere: A Simple, Fast, and Reliable Protocol for In‐house AAV Vector Production Based on Chloroform Extraction

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