Systemic AAV vectors for widespread and
targeted gene delivery in rodents
Rosemary C. Challis
1,3
, Sripriya Ravindra Kumar
1,3
, Ken Y. Chan
1
, Collin Challis
1
, Keith Beadle
1
,
Min J. Jang
1
, Hyun Min Kim
1
, Pradeep S. Rajendran
2
, John D. Tompkins
2
, Kalyanam Shivkumar
2
,
Benjamin E. Deverman
1
and Viviana Gradinaru
1
*
In the format provided by the authors and unedited.
1
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.
2
Cardiac Arrhythmia Center and
Neurocardiology Research Center of Excellence, University of California, Los Angeles, Los Angeles, CA, USA.
3
These authors contributed equally:
Rosemary C. Challis and Sripriya Ravindra Kumar. *e-mail: viviana@caltech.edu
NATURE PROTOCOLS
|
www.nature.com/nprot
PROTOCOL
https://doi.org/10.1038/s41596-018-0097-3
1234567890():,;
1234567890():,;
Supplementary Table 1. Use of AAV
-PHP
capsids for efficient transduction
across
specific organs and cell populations.
Species
/strains
, organs, and cell populations examined
to-date following intravenous administration of AAV
-PHP viruses. To restrict gene expression to
distinct cell types, use rAAV genomes with cell type-specific gene regulatory elements and/or
Cre
- or Flp-dependent recombination schemes
(
Figs. 2-4
and
Table 1
).
Neurons and astrocytes in
cortical spheroids
Ref. 2
Good
AAV-PHP.B
Brain and spinal cord
Ref. 3
Ref. 4
Rat (Sprague-Dawley), P1 and
6 weeks
Neurons throughout the brain
and spinal cord
Organs/cell populations
transduced
References
Mouse (C57BL/6J and Cre
driver lines),
≥
6 weeks
Neurons, astrocytes,
oligodendrocytes, and
endothelial cells across all brain
and spinal cord regions
examined. Transduction in the
retina occurs with variable
efficiency across all layers
Ref. 1-3
AAV-PHP.eB
Good
AAV-PHP capsid
Production
Species (strain or line), age
injected
Mouse (C57BL/6J), P0-P1
Human iPSCs
Mouse (C57BL/6J,
C57BL/6NCrl, FVB/NCrl,
129S1/SvImJ, 129T2/SvEmsJ,
and Cre driver lines),
≥
6 weeks
Rat (Fischer), 6 weeks
Rat (Long Evans), 4 weeks
Rat (Sprague-Dawley), 6 weeks
AAV-PHP.A
Poor
Mouse (C57BL/6J), 6 weeks
Astrocytes throughout the brain
Sensory neurons, peripheral
ganglia (sympathetic, nodose,
dorsal root, and cardiac
ganglia), and the myenteric and
submucosal plexus of the
enteric nervous system. Robust
transduction of heart muscle,
skeletal muscle, and circular
and longitudinal muscle of the
digestive tract, as well as the
liver and lungs
AAV-PHP.S
Good
Ref. 5, this work
Mouse (C57BL/6J and Cre
driver lines), 6-8 weeks
Ref. 2
Similar to AAV-PHP.B but with
increased efficiency of neuronal
transduction
Ref. 5, this work
Ref. 6
Supplementary Table 2. Transfection calculator.
This is an interactive calculator and
provided as an Excel fil
e (see Step 2 and REAGENT SETUP).
Supplementary Table 3. Pouring the iodixanol
density step solutions.
Determine the
number of gradients needed and prepare the iodixanol density step solutions (see REAGENT
SETUP). The 15% step contains high salt to destabilize ionic interactions between viral particles
and cell proteins in the clari
fied lysate
7
. In Step 16 (option B), prepare more step solution
than is
needed (see REAGENT SETUP).
Supplementary Table 4. Titration calculator.
This is an interactive calculator and provided as
an Excel file
(see Step 42 and REAGENT SETUP)
.
SUPPLEMENTARY REFERENCES
1
Allen, W. E.
et al.
Global Representations of Goal
-Directed Behavior in Distinct Cell
Types of Mouse Neocortex.
Neuron
94
, 891-907, doi:10.1016/j.neuron.2017.04.017
(2017).
2
Deverman, B. E.
et al.
Cre
-dependent selection yields AAV variants for widespread gene
transfer to the adult brain.
Nature Biotechnology
34
, 204-209, doi:10.1038/nbt.3440
(2016).
3
Morabito, G.
et al.
AAV-PHP.B-
Mediated Global
-Scale Expression in the Mouse Nervous
System Enabl
es GBA1 Gene Therapy for Wide Protection from Synucleinopathy.
Molecular Therapy
25
, 2727-2742, doi:10.1016/j.ymthe.2017.08.004 (2017).
4
Jackson, K. L., Dayton, R. D., Deverman, B. E. & Klein, R. L. Better Targeting, Better
Efficiency for Wide-Scale Neur
onal Transduction with the Synapsin Promoter and AAV
-
PHP.B.
Frontiers in Molecular Neuroscience
9
, doi:10.3389/fnmol.2016.00116 (2016).
%
Solution (ml)
1
2
4
6
8
DPBS + high salt
5.0
9.9
19.8
29.7
39.6
60% iodixanol
1.7
3.3
6.6
9.9
13.2
DPBS + low salt
3.9
7.7
15.4
23.1
30.8
60% iodixanol
2.8
5.5
11.0
16.5
22.0
Phenol red
0.1
0.1
0.2
0.3
0.4
DPBS + low salt
1.8
3.7
7.3
11.0
14.7
60% iodixanol
3.7
7.3
14.7
22.0
29.3
60% iodixanol
5.5
11.0
22.0
33.0
44.0
Phenol red
0.1
0.1
0.2
0.3
0.4
Number of gradients
Step
Volume of each step
per gradient (ml)
40
60
6
6
5
5
15
25
5
Chan, K. Y.
et al.
Engineered AAVs for efficient noninvasive gene delivery to the central
and peripheral nervous systems.
Nature Neuroscience
20
, 1172-1179,
doi:10.1038/nn.4593 (2017).
6
Dayton, R. D., Grames, M. S. & Klein, R. L. More expansive gene transfer to the rat
CNS: AAV PHP.EB vector dose
-response and comparison to AAV PHP.B.
Gene
Ther
apy
, doi:10.1038/s41434-018-0028-5 (2018).
7
Zolotukhin, S.
et al.
Recombinant adeno-associated virus purification using novel
methods improves infectious titer and yield.
Gene Therapy
6
, 973-985,
doi:10.1038/sj.gt.3300938 (1999).