Databinge AAV Presentation · Plasmid DNA •Plasmid = double-stranded DNA that can replicate...

Databinge AAV PresentationMatthieu Vanni + Alexa Nelson

Table of Contents

Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Addgene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Transfection and Transduction . . . . . . . . . . . . 5 - 9 AAVs General Info . . . . . . . . . . . . . . . . . . . . . 10 -12 AAV Serotypes . . . . . . . . . . . . . . . . . . . . . . . . 13 - 18 AAV Promoters . . . . . . . . . . . . . . . . . . . . . . . 19 - 36 Buying AAVs . . . . . . . . . . . . . . . . . . . . . . . . . . 37 - 38 AAV Biosafety . . . . . . . . . . . . . . . . . . . . . . . . 39 - 40 Handling AAVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Animal Care . . . . . . . . . . . . . . . . . . . . . . . . . . 42 - 43 Rat Stereotaxic Surgery for AAV Injection . 44 - 59 Animal Resource Unit + Follow-up . . . . . . . 60 - 62 Image Examples in Brain Slices . . . . . . . . . . 63 - 68 Image Examples in vivo . . . . . . . . . . . . . . . . 69 – 76 Additional Links . . . . . .. . . . . . . . . . . . . . . . . . . . . 77

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Plasmid DNA

• Plasmid = double-stranded DNA that can replicate independently of chromosomal DNA

• Found in bacteria

• 1,000 – 20,000 base pairs (1-20 kbp)

• Used to code for probes like GCaMP

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https://www.addgene.org/mol-bio-reference/plasmid-background/

Addgene

• Non-profit plasmid repository • https://www.addgene.org• Plasmids stored in bacteria in stabs (what is mailed)• Great resource for information about molecular biology, viruses, etc.• Downloadable Molecular Biology Reference

• Navigating the website:1. Search

• Eg. mitochondrial calcium

2. Sort + Filters to narrow down• Eg. popularity + mammalian expression

3. Select plasmid• Eg. CMV-mito-R-GECO1 (#46021)

4. Literature Search• Zhao Y, Araki S, Wu J, Teramoto T, Chang Y-F, Nakano M, et al (2013) An

expanded palette of genetically encoded Ca2+ indicators. Science 333: 1888-1891.

https://www.ncbi.nlm.nih.gov/pubmed/?term=219037794

https://www.addgene.org/recipient-instructions/streak-plate/

Transfect or Transduce Plasmid

1. Isolate plasmid DNA (pDNA) from Addgene bacterial stab• Eg. with QIAGEN midiprep kit• https://www.qiagen.com/us/?akamai-feo=off

2. Transfect DNA:• Lipofection

• (eg. Lipofectamine [Invitrogen], Effectene [Qiagen], Lipid Nanoparticles)• Electroporation• Microinjection• Biolistics

3. OR, transduce DNA using viruses:• Package DNA in viruses• Purchase pre-packaged from provider like Laval or UPenn

• Cheap

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Isolate pDNA

Plasmid from Addgene

Transfect Transduce(eg. in mammalian cultures, in vivo)

Transfection

• Advantages:• Simple + fast• Less biosafety/biohazard paperwork

• Disadvantages:• Can be less efficient (eg. with lipofection)

• Examples of reagents:• Lipofectamine (Thermofisher)http://www.thermofisher.com/ca/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html

• Lipid Nanoparticles (LNPs):http://www.liposomes.ca• Dr. Pieter Cullis @ UBC

• Packaging size limit?

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Example of transfection protocol(Thermo Fisher Scientific)

Transduction with Viruses

• Advantages:• Efficient

• Disadvantages• More paperwork

• Packaging size limits

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Transduction

https://www.addgene.org/viral-vectors/

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Summary of Transduction and Transfection Methods in Neurons

• Karra D, Dahm R (2010) Transfection techniques for neuronal cells. 30(18): 6171-6177.

https://www.ncbi.nlm.nih.gov/pubmed/?term=20445041

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Adeno-Associated Viruses (AAVs)

• Wild-type AAV: • Parvovirus not associated with pathology• ~ 20 nm• ssDNA ~4.7 kb• Genome:

• 2 inverted terminal repeats (ITRs) • 2 open reading frames (ORFs)

• Rep (4 proteins in replication) = Rep78, Rep68, Rep52, Rep40 • Cap (3 proteins in capsid formation) = VP1, VP2, VP3 (virion particle)

• Integrates into AAVS1 on human chromosome 19

• Good resources for information:• https://www.addgene.org/viral-vectors/aav/aav-guide/• http://ppn.med.sc.edu/Adeno-associated.Virus.asp• http://vectorbiolabs.com/vbs/page.html?m=281

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AAVs in Research

• In research use recombinant AAV (rAAV) depleted of rep and cap genes • Virus does not replicate• Only leaves ITRs around gene of

interest

• Maintained extrachromosomally

• Many serotypes

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http://ppn.med.sc.edu/Adeno-associated.Virus.asp

• Produce recombinant AAVs by co-transfecting 3 plasmids in cell line:

1. rAAV vector (gene of interest with ITRs)

2. AAV replication and cap genes

3. Helper genes

• Isolate virus

• Many companies do this, cheap to buy

Recombinant AAVs (rAAVs)

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AAV Serotypes

• AAV2 one of most widely used serotypes but low transduction efficiency

• AAV Pseudotyping• AAV2 genomes in capsids from other AAVs• Eg. rAAV2/5

• Genome based on AAV2, capsid based on AAV5• Tropism should be same as AAV5

• AAV2/5 more widely distributed in brain, better transduction efficiency than AAV2/2

• Multiple serotypes, eg. AAV-DJ hybrid capsid from eight serotypes • Higher transduction efficiency compared to wt

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Addgene

AAV Serotypes

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UPenn

AAV Serotypes

?Wide expression

Retrograde transport

So which serotype to use?

1. Read the literature to see which serotype would be optimal• Aschauer DF, Kreuz S, Rumpel S (2013) Analysis of transduction efficiency, tropism and axonal

transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. Plos One 8: 1-16.

https://www.ncbi.nlm.nih.gov/pubmed/?term=24086725

• Watakabe A, Ohtsuka M, Kinoshita M, Takaji M, Isa K, Mizukami H et al (2015) Comparitiveanalyses of adeno-associated viral vector serotypes 1, 2, 5, 8 and 9 in marmoset, mouse and macaque cerebral cortex. Neuroscience Research 93: 144-157.

https://www.ncbi.nlm.nih.gov/pubmed/?term=25240284

2. Test for yourself what is optimal in your system• GFP-expressing AAV2 serotypes from Harvard at low cost

• https://vectorcore.mgh.harvard.edu/AAV_guide

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Aschauer DF et al. (2013) Paper

17Expression Inflammatory Response

AAV 2/9 for Brain

• AAV9 has good spread

• But depends on what the study question is • eg. might not be appropriate if doing tracing

• AAV2/9 recommended by Harvard for brain

• Wade Regehr (Hosted by Dr. Ann Marie Craig)• AAV2/9 with Synapsin promoter• Jackman SL, Turecek J, Belinsky JE, Regehr WG (2016) The calcium sensor

synaptotagmin 7 is required for synaptic facilitation. Nature 529: 88-91. https://www.ncbi.nlm.nih.gov/pubmed/?term=26738595

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AAV1.Syn.flex.ChR2-GFP.WPRE.SV40

“.” dot to separate sequences“-” dash for fusion proteinsserotype

Sequences of interest(will be transcripted/translated to the precious protein wanted)

Associated sequences(won’t be expressed but will impact the expression of the sequences of interest)

Promoter

Extrinsic Specificity(Optional)

AAV1.Syn.cre

AAV Grammar

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Promoters (Mammalian Applications)• General expression

• CMV: Cytomegalovirus• CBA (emCBA: enhance mini): chicken β actin (CI: CBA intron?)

• CMV enhancer + CBA = CAG (or CB7) synthetic promoter, CAGGS(?)

• EF1a: Human elongation factor-1 alpha. • PGK1: phosphoglycerate kinase• Ubc: ubiquitin C

• Specific expression• Syn (hSyn, hSyn1): synapsin promoter every neurons• CaMKIIa excitatory neurons• hThy1 excitatory neurons• GFAP (Glial fibrillary acidic protein) glial cells

• GfaABC1D or GFA104 (0.7kb): compact GFAP (2.2kb)

• TH: Tyrosine hydroxylase dopamine cells• MCK: muscle creatine kinase myocytes • cTNT: Chicken Cardiac Troponin T cardiomyocytes • TBG: Thyroxine-binding globulin hepatocytes

• Tetracycline dependant• TRE: tetracycline transactivator (tTA) protein (Tet-ON/Tet-Off system)

20https://www.addgene.org/tetracycline/

CaMK2a-tTA mouse(jax 003010)

Other Sequences

• WPRE: (Woodchuck Hepatitis Virus) Posttranscriptional Regulatory Element. Enhance the expression.

• poly A sequences (SV40, hGH, BGH, rBG, NRN …): Motif AAUAAA which promotes both polyadenylation and termination. Define the end of a transcription.• e.g. AAV1.Syn.GCaMP6s.WPRE.SV40

• WGA (wheat germ agglutinin) transported trans-synaptically used as a circuitry tracer.

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3651700/

Multicistronic Vectors

• IRES Elements• poliovirus RNA, 600b!

• Multiple translation site

• 2A (x2A) sequence (e.g. P2A, GSG.P2A)• foot-and-mouth disease virus, super short

• self-cleaving

• e.g. AAV1.Syn.GCaMP6s.P2A.NES-jrGECO1a

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Common gene of interest and fusion proteins/sequence

• Functional Neurophotonics and other genetic engineering• Calcium sensors : GCaMP (6s, 6m, 6f, 3), jrGECO1a, jRCaMP1a, CaMPARI, Twitch2B• Glutamate sensor : iGluSnFR• Voltage sensor: ArcLight• Optogenetic inhibitor: Jaws, Arch, NpHR, …• Optogenetics activator: ChR2 (many), ChETA, Chronos, ChrimsonR, C1V1• Pharmacogenetics (DREADD): hM3D, hM4D• Diphtheria toxin A fragment: dtA

• Immunohistochemistry tag:• HA: hemagluttinin (e.g. HA-hM4D)

• Trafficing• NES, H2B, NLS: nuclear localization sequence (fusion protein, e.g. NES-jrGECO1a)• Lck, ER2, KGC, TS, Myr: membrane localization (e.g. Lck-GCaMP6)

• Fluorescent fusion proteins:

GFP, YFP, (e.g. ChR2-GFP)

mCherry, tdTomato, dsRed, …

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Cre/Lox RecombinationCre recombinase recombine LoxP sites (34b)

• Cre, creERT2 (Tamoxyfen inductible), cre-GFP• e.g. AAV1.Syn.cre

Cre-dependant strategies:

• gene knock-out: “floxing”, for “flanked by loxP”• LoxP – x – LoxP

• Cre-dependant gene expression: “lox-stop-lox” or “LSL” cassette • e.g. AAV1.CAG.LSL.tdTomato.bGH

• flex (“flip-excision”) or DIO (Double-floxed Inverse Orientatio) or or double flox (dflox)• flex-frwd: cre inactivation by inversion• e.g. AAV1.Syn.Flex.GCaMP6f.WPRE.SV40

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CAG stop tdTomato

CAG tdTomatostop

LSL:

Cre/Lox Alternative and Association

• Flippase Flp vs. FRT site• e.g. AAV-Syn-FLPo

• Dre recombinase vs. rox site

• Combining the cre/lox, flp/frt and dre/rox: INTRSECT (INTronicRecombinase Sites Enabling Combinatorial Targeting, Fenno et al. 2014 Nature Methods): Bolean logic• e.g.• AAV-nEF-Con/Fon hChR2(H134R)-EYFP-WPRE• AAV-nEF-Con/Foff hChR2(H134R)-EYFP-WPRE• AAV-nEF-Coff/Fon hChR2(H134R)-EYFP-WPRE

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Where to buy AAVs?

• Canadian Neurophotonic Plateform (Univ. Laval): http://neurophotonics.ca/molecular-tools-platform

• Upenn (Univ. of Pennsylvania): http://www.med.upenn.edu/gtp/vectorcore/• Vector Biolabs http://www.vectorbiolabs.com/Recombinant-AAV/ ?

• UNC (Univ. of North Carolina): http://www.med.unc.edu/genetherapy/vectorcore

Never tried:

• Stanford: http://med.stanford.edu/gvvc/stockviruses.html

• Duke: https://sites.duke.edu/dvvc/collection-aav/

• Addgene (Cambridge, MA): http://www.addgene.org/viral-service/aav-prep/

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Serotypes mostly 1, 5, 9Synapsin promoter

CaMPARI: Switchable GCaMP

WPRE: enhancer

polyA sequence = stop

Different GCaMP

CAG promoter

CaMK2 promoter

NES: nuclear localization

jrGECO1a: red GCaMP6

jRCaMP1a: red GCaMP6

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iGluSnFR: glutamate sensor

GFAP promoter

Twitch2B: FRET calcium indicator

mRuby2: red reporter

2A: self-cleaving GfaABC1D GFAP promoter

Lck=membrane localization

Jaws-KGC: red activated chloride pump targeted to membrane

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hGH,bGH polyA

tdTomato, GFP fusion protein

29EF1a promoter

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Flex inversion

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gene knock-out: “floxing”

2A cleavage

DIO=flex

CBA promoter

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Cre express under CMV promoter

GFP-cre

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Neuron-Specific AAV Promoter

• Synapsin 1 good for general neuronal expression• Hioki H, Nakamura H, Okunomiya T, Ohira K, Nakamura K, Kuroda M et al (2007) Efficient

gene transduction of neurons by lentivirus with enhanced neuron-specific promoters. Gene Therapy 14: 872-882.

https://www.ncbi.nlm.nih.gov/pubmed/?term=17361216

• Kugler S, Kilic E, Bahr M (2003) Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area. Gene Therapy 10: 337-347.

https://www.ncbi.nlm.nih.gov/pubmed/?term=12595892

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Buying AAVs

• Need to fill out MTA forms (Material Transfer Agreement)• https://uilo.ubc.ca/researchers/forms-and-templates

• Example of cost:• From Laval with internal pricing 100 μl aliquot = $200

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BUT… before you buy/receive AAVs

• Need in place:1. Biosafety Certificate (with Biohazard Assessment)

2. Animal Care Protocol for in vivo work

• These applications are completed on RISe (Research Information Services)• https://www.rise.ubc.ca

• Might be amendment or new application

• Use other labs’ protocols as templates• We (MacVicar lab) based ours off of Snutch protocol (thank you!)

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1. Biosafety Certificate

https://ors.ubc.ca/compliance-reporting/compliance-requirements/biosafety

• What’s included:1) Study team 2) Funding sources3) Procedures and Methodology 4) Recombinant Viral Vectors 5) Ethics

• Possibly add as amendment

• Also need Biohazard Risk Assessment (component of Biosafety Certificate)

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Biosafety for Viruses

• CL = containment Level

• RG = Risk Group

• AAVs:• CL1 (RG1) • Advantageous, less paperwork/equipment

• Lentivirus:• Mainly CL2 (RG3)• Or CL2 with CL3 Procedures

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Handling AAVs

• Keep AAVs on ice• Aliquot in Class 2 Biosafety Hood

• Small volumes (eg. 5 μl in Eppendorfs, avoid repeated freezing-thawing)• Can be freeze-thawed several times without loss in activity but should avoid • Stanford Vector Core – Care and Handling of AAVs:

http://med.stanford.edu/gvvc/AAV.html

• Store at -80 oC• Dispose of AAV + related material (eg. pipette tips) in 10 % bleach > 30 min

(in beaker)• When handling PPE

• N95 respirator• Gloves• Lab coat

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Animal Care Protocol for in vivo work

• What’s included:1) Study team 2) Study Dates and Funding 3) Animal Information & Type of Animal Review4) Animal Information, Procedures, Justification5) Animal Monitoring6) Drugs and Chemicals

• Vet viewing/approval for members performing procedure

• Animal Care Committee meets once a month, can take a while to get new application submitted: • https://animalcare.ubc.ca/animal-care-committee/process/submission-deadlines• But once application approved, can make amendments (faster)

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UBC Animal Care’s SOPs

• Cite UBC Standard Operating Procedures (SOPs):• https://animalcare.ubc.ca/planning-your-research/sops-guidelines• Relevant SOPs for stereotaxic surgery and injections (rats):

• Rat Stereotaxic Surgery (was not able to find online, e-mail Animal Care Committee for copy)• Rodent Survival Surgery (SOP ACC-02-2016)• Rodent Anesthesia (SOP ACC-01-2015)• Subcutaneous injection in mice and rats (TECH 11)• Local Anesthetic SOP (TECH 16)• Ketoprofen SOP (TECH 18)• SOP Euthanasia of Adult Rodents using CO2

• Monitoring sheets• SOP 017 – Monitoring Policy

• https://animalcare.ubc.ca/animal-care-committee/policies-and-guidelines/017-animal-monitoring-sheets-samples

• Grading system found online

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Rat Stereotaxic Surgery for Viral Injection

• UBC Rat Stereotaxic Surgery SOP • New

• Supposed to be available online, but needed to e-mail Animal Care Committee for copy

• Don’t know if there’s a Mouse Stereotaxic SOP

• Cetin A, Komai S, Eliava M, Seeburg PH, Osten P (2007) Stereotaxic gene delivery in the rodent brain. Nature Protocols 1: 3166-3173.

https://www.ncbi.nlm.nih.gov/pubmed/17406580

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