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. 2016 Oct 19;92(2):372-382.
doi: 10.1016/j.neuron.2016.09.021. Epub 2016 Oct 6.

A Designer AAV Variant Permits Efficient Retrograde Access to Projection Neurons

D Gowanlock R Tervo  1 , Bum-Yeol Hwang  2 , Sarada Viswanathan  1 , Thomas Gaj  2 , Maria Lavzin  3 , Kimberly D Ritola  1 , Sarah Lindo  1 , Susan Michael  1 , Elena Kuleshova  4 , David Ojala  2 , Cheng-Chiu Huang  1 , Charles R Gerfen  5 , Jackie Schiller  3 , Joshua T Dudman  1 , Adam W Hantman  1 , Loren L Looger  1 , David V Schaffer  6 , Alla Y Karpova  7
Affiliations

A Designer AAV Variant Permits Efficient Retrograde Access to Projection Neurons

D Gowanlock R Tervo et al. Neuron. .

Abstract

Efficient retrograde access to projection neurons for the delivery of sensors and effectors constitutes an important and enabling capability for neural circuit dissection. Such an approach would also be useful for gene therapy, including the treatment of neurodegenerative disorders characterized by pathological spread through functionally connected and highly distributed networks. Viral vectors, in particular, are powerful gene delivery vehicles for the nervous system, but all available tools suffer from inefficient retrograde transport or limited clinical potential. To address this need, we applied in vivo directed evolution to engineer potent retrograde functionality into the capsid of adeno-associated virus (AAV), a vector that has shown promise in neuroscience research and the clinic. A newly evolved variant, rAAV2-retro, permits robust retrograde access to projection neurons with efficiency comparable to classical synthetic retrograde tracers and enables sufficient sensor/effector expression for functional circuit interrogation and in vivo genome editing in targeted neuronal populations. VIDEO ABSTRACT.

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Figures

Figure 1
Figure 1. Directed evolution of rAAV2-retro
(A) Schematic of the directed evolution procedure. Plasmid libraries containing variant AAV cap genes previously generated by error prone PCR, peptide insertion, randomization of loop regions, and DNA shuffling were packaged, and the resulting viral vector libraries were injected into substantia nigra or cerebellar cortex. Three weeks later, striatal or hindbrain, respectively, tissues were removed, viral genomes isolated, and selected cap genes amplified and packaged for the next round of selection.(B) Example retrograde labeling of projection neurons with the final chosen variant, rAAV2-retro. Retrograde access to intermingled sub-populations of neurons in various brain regions projecting to three striatal compartments was assessed two weeks after delivery of rAAV2-retro carrying different fluorescent proteins into the corresponding axonal fields. rAAV2-retro-tdTomato was injected into dorsolateral striatum (DLS), rAAV2-retro Ruby2_FLAG into dorsomedial striatum (DMS), and rAAV2-retro EGFP into ventrolateral striatum (VLS). Unlike in subsequent experiments (see Figures 2-6), the three probes were visualized by immunohistochemistry to ensure robust detection of retrograde transport at this early time point. Ctx: cortex; BLA: basolateral amygdala; IL TH: intralaminar thalamic nuclei; CL: centrolateral thalamic nucleus; CM: centromedial thalamic nucleus. Scale bar: 1mm. See also Figures S1 and S4, and Movie S1.
Figure 2
Figure 2. Quantification of retrograde transport efficiency
(A) Efficient labeling of the corticopontine tract throughout the rostro-caudal axis via basal pontine injection of rAAV2-retro-DIO-CAG-tdTomato in a layer V-specific Cre mouse line (Rbp4_KL100 Cre). Top panel: schematic of the experiment. Injection site was marked by co-injecting rAAV1-CAG-EGFP. BPN: Basal pontine nuclei. Bottom panel: unamplified tdTomato and EGFP expression three weeks after injection. Scale bar: 1 mm. (B) Quantification assay design. Top panel: schematic of the experiment. Corticopontine labeling was assessed in sagittal sections lateral to the injection tract (∼1 mm lateral with respect to midline) taken from Rosa26-Lox-STOP-Lox-H2B-EGFP animals injected in the BPN with various AAV serotypes carrying Cre recombinase transgene. Arrow indicates expected nuclear GFP labeling in cortical neurons of the corticopontine tract. Middle panel: representative image of an rAAV2-injected brain. Bottom panel: representative image of rAAV2-retro-injected brain. Scale bar for both the panels: 1 mm. (C) Schematic of the semi-automated quantification procedure. Fluorescent nuclei (green) were automatically detected and counted along a manually drawn line that traced the length of cortical layer V (black). (D) Retrograde transport efficiency for different AAV serotypes and for canine adenovirus type 2 (CAV-2) (See Experimental Procedures). Error bars represent the S.E.M. See also Figures S2 and S3.
Figure 3
Figure 3. Generality of retrograde transport afforded by rAAV2-retro
(A) Representative images showing extensive labeling in the main input structures to the dorsal striatum including cortex (panel 1), amygdala (panel 3), and thalamus (panel 4). Scale bars: 800 micrometers for panels 1 and 2,200 micrometers for panels 3 and 4.Ctx: cortex; CP: caudate/putamen; BLA: basolateral amygdala; Th: thalamus; (B) Schematic of automated whole-brain quantification of retrograde labeling. Brains of Rosa26-LSL-H2B-EGFP injected with rAAV2-retro-hSyn1-Cre were imaged to visualize DAPI stained nuclei and green fluorescence from H2B-GFP expressing nuclei. The green channel was used to detect labeled neurons; the blue channel was aligned to the Nissl images from the Allen Brain Institute's standard mouse brain (see Experimental Procedures). The alignment permitted detected neurons to be assigned to different regions using the annotation provided by the brain atlas. Scale bar: 1.25 mm. (C) Whole-brain quantification of retrograde labeling out of a small region of the dorsomedial striatum. Abbreviations for the different brain areas are given according to the Allen Brain Atlas. Arrow highlights the SNc. Error bars represent the S.E.M.
Figure 4
Figure 4. Combining rAAV2-retro system with Cre driver lines for selective access to parallel corticostriatal pathways
(A) Schematic of the experiment. rAAV2-retro carrying a Cre-dependent color flipping fluorescent reporter was injected into the striatum of a cortical layer V-specific Cre line. (B) Two corticostriatal pathways differentially labeled through a Cre-dependent inversion of the reporter in one (layer V), but not the other (layer II/III), pathway. A presumed Cre-independent inversion— typical for all AAV vectors—was observed in a small fraction of corticostriatal neurons in layer II/III (white arrows). Scale bar: 50 micrometers.
Figure 5
Figure 5. rAAV2-retro supports sufficient transgene expression for functional circuit interrogation
(A) Schematic of the experiment. Expression of calcium indicator GCaMP6f is restricted to corticopontine neurons using localized injection of rAAV2-retro into the BPN. (B) Cross section of the mouse brain showing GCaMP6f expression throughout the corticopontine tract. Scale bar: 50 micrometers. (C) Maximum projection of an in vivo two-photon calcium image taken during a single reach showing layer V-pyramidal tract somas and apical dendrites. Scale bar: 50 micrometers. (D) Activity of 89 ROIs during a single paw reach repetition (broken line denotes the tone “go” signal). (E-F) Two examples of single corticopontine neurons during 40 consecutive trials (same animal as in B-D). Scale bar: 4 seconds.
Figure 6
Figure 6. rAAV2-retro system enables in vivo genome editing using CRISPR/Cas9
(A) Schematic of the experiment. Top panel: The rAAV2-retro system was used to deliver Staphylococcus aureus Cas9 (SaCas9) single guide RNA combination engineered to ablate expression of tdTomato. Bottom panel: rAAV2-retro carrying the SaCas9-anti-tdTomato payload was injected into the BPN of mice expressing tdTomato from a single genomic locus in layer V neurons. SaCas9 was epitope tagged, permitting identification of retrogradely labeled neurons (green channel). Upward arrows: expected labeling following successful ablation of tdTomato. Downward arrows: expected labeling if tdTomato expression is unaffected. (B) Representative images from brain sections of animals that received the CRISPR/Cas9 system targeted against tdTomato or carrying a non-targeted guide. tdT: tdTomato. Scale bar: 50 micrometers. (C) Efficiency of ablation. Error bars represent the S.E.M.
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