Dr. Randy Prather - Porcine Reproductive and Respiratory Syndrome Virus Resistant Pigs

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Transcript of Dr. Randy Prather - Porcine Reproductive and Respiratory Syndrome Virus Resistant Pigs

CD163

The Role of CD163 and SIGLEC1 in Sensitivity to Porcine Reproductive and Respiratory Syndrome Virus Infection

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Genetic Engineering of Pigs at MissouriCancer (CAG-Floxed-Stop, KRASG12D, p53R167H; caCSN2-miBRCA1-RASSF1)Cardiovascular Disease (hFAT-1+; TEK-NOS3+; TEK-CAT+)Cardiac Development (Floxed FGF8)Cystic Fibrosis (CFTR-/-; CFTRF508/F508; CFTRF508/F508, Tet-on CFTR+; CFTRF508/F508, FABP2-CFTR+; shRNA CFTR)Diabetes (ssGIP-hINS+)Disease Resistance (SIGLEC1-/-; CD163-/-; CD163-/domain 5; CD163-/Domain 5 swap with domain 8 of hCD163L; CD163-/-, SIGLEC1-/-; CD1D-/-)Enhancing Meat Quality (hFAT1+)Muscular Dystrophy (DMD+/exon 46)Pharmaceuticals (FIX+, VWF+, SERPINA1+; FVIII+, SERPINA1+, FURIN+)Regenerative Medicine (IL2RG+/-; RAG2-/-)Retinitis Pigmentosa (RHO P23H)Spinal Muscular Atrophy (SMN+/-; SMN+/-, hSMN2+)Suicide Prodrug for Liver Transplantation (ALB-TK1+; AFP-CDA+) Tracking/Tools (CAG-eGFP+; CMV-eGFP+; ZP3-CRE; UBC-Tomato+; CAG-Tomato+; UBC-nls eGFP+; PSMA1-eGFP fusion protein +)Xenotransplantation (GGTA1-/-; GGTA1-/-, CD55+; GGTA1CD55/+; GGTA1-/-, CD55+, CD59+, ENTPD1+, THBD+; SIGLEC1-/-)Plus others that I cant talk about- over 45 different modifications and over 1,000 cloned pigs.

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OutlineModel of Infection for PRRSvSIGLEC1CD163Timelines & From Here to Where?

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PRRSv Infectivity Was Thought to Result from 3 Specific Entry MediatorsInitial binding with heparan sulfateBinding/Internalization by SialoadhesinInternalization/uncoating of the virus by CD163

Van Breedam et al., 2010

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Heparan Sulphate Binding

Binding and Internalization by Sialoadhesin(SIGLEC1)

Uncoating of the virus by CD163

Genome Release in endosomes dependent on low pHVan Breedam et al., 2010

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Organization the Sialoadhesin Gene and Targeting Vector Design.

Southern Probe

Prather et al 13 J Virology

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A targeted clone (4-18) was identified and used both for somatic cell nuclear transfer and to propagate for Southern blot analysis.Screened >2,000 colonies: efficiency ~7.0 x 106

TransfectionPrather et al 13 J Virology

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Enucleation

Fusion or Injection and ActivationDevelopment

Transfect or Transduce and Select

Synchronize?

Nuclear Transfer

US Patent #6,211,429

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SIGLEC1+/- pigsBorn 1/16-18/2011

Patent PendingPrather et al 13 J Virology

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First SIGLEC1 -/- Pigs (F2 pigs) BornOctober 18, 2012

4 SIGLEC1 -/-4 SIGLEC1 +/-4 SIGLEC1 +/+ Wild TypePrather et al 13 J Virology

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PRRSv ChallengePiglets were shipped to Kansas State UniversityAfter 1 week acclimationLow passage PRSSV isolate (KS-06: North American isolate collected in 2006)105 TCID50 of virus diluted in 3 mL intramuscularly intranasallyBlood sampled on days 0, 4, 7, 14, 21, 28 & 35.Prather et al 13 J Virology

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Clinical OutcomeAssessed daily for clinical signsPigs co-housed No apparent clinical signs before infectionDay 3 after infection all pigs began to exhibit mild respiratory signs (sneezing, coughing, increased breathing)These resolved within 1- to 2-weeks. In a single WT pig more severe signs reappeared on day 28.Euthanized at the end of the studyHistology- Lungs- no difference as all had moderate to severe multifocal interstitial pneumonia.Collected alveolar macrophages. Prather et al 13 J Virology

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Do the SIGLEC1-/- pigs express SIGLEC1?

Flow Cytometry on porcine alveolar macrophages at Kansas State University- Bob Rowland.CD163SIGLEC1Wild-Type pig2nd AbPrather et al 13 J Virology

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CD163SIGLEC1Wild-Type pig2nd AbDo the SIGLEC1-/- pigs express SIGLEC1 in PAMs?SIGLEC1-/- pig

CD163SIGLEC12nd AbPrather et al 13 J Virology

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Prather et al 13 J Virology

SIGLEC1

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Virus in blood that killed cells in vitro- cytopathic effect.

Prather et al 13 J Virology

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Viral RNA/nuclei acid

Prather et al 13 J Virology

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TimelineConceive Idea June 02.Search for $$$Founder male heterozygotes Born Jan 11.Founders reach puberty Oct-Sept 11.F1s born (heterozygous males and females) Nov 11-Jan 12.F1s reach puberty summer 12.Homozygous Knockout animals born Oct 12.Challenge experiments began Nov 12.Publication 13 almost 11 years later.

Prather et al 13 J Virology

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SIGLEC1 ConclusionsSIGLEC1-/- is not embryonic lethal.Potential problem- KO mice have subtle changes in T- and B-cell populations, and reduced immunoglobulin M (Oetke et al 06).SIGLEC1-/- may have altered IgG response, but variation was high and sample size was small.SIGLEC1-/- did not affect susceptibility to PRRSv.Could cells from these pigs be useful diagnostic tools?

Prather et al 13 J Virology

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The Case for CD163A series of experiments similar to that for SIGLEC1 provided evidence that CD163 is not only an entry mediator but also facilitates infection (as outline by Welsh & Calvert 10). Other candidates include CD151, VIM and CD209.

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CD163 CD163 is a member of the scavenger receptor cysteine-rich (SRCR) superfamily

17 exons, 16 introns

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Determine which extracellular domains were required for PRRSV infectivityHEK293T were transfected with SIGLEC1 and CD163 deletion constructsCells were inoculated with PRRSv for 24 hr and fixedNumber of infected cells were counted

Deletion Constructs

Van Gorp et al 2010 J Vir

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Chimeric MutantsCD163 is a member of gene familyhCD163L (CD163-like)12 extracellular domainsDoes not function in infectivity

Swap domains from hCD163-like and CD163 and determine infectivity

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Van Gorp et al 2010 J VirHuman CD163

Human CD163L

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Two Proposed Targeting ApproachesTraditional knockout by homologous recombination and addition of a stop codonDomain SwapRemove extracellular domain SRCR5 from CD163Replace with extracellular domain 8 from hCD163L

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Haptoglobin-Hemaglobin SRCR #3Erythroblast SRCR #2TNFSF12 (aka TWEAK) SRCR #1-4 & #6-9 Bacteria SRCR #2PRRSv- SRCR #5

Advantages of a Domain Swap

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But is a Domain Swap Necessary?Haptoglobins ability to promote binding of hemoglobin to CD163 in human has high affinity, but not in mice.In mice CD163 accounts for a small part of hemoglobin clearance.Knockout of CD163 results in No apparent phenotype change and offspring were viable and fertile. Etzerodt et al 12. Antioxidants & Redox Signaling, DOI: 10.1089/ars.2012.4605

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CRISPR-Mediated Gene EditingNon-Homologous End JoiningCan result in changing a handful of base pairsThus can remove exons/domains or knockout a gene.Swine genome is ~2,700,000,000 base pairs.

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CD163 Timeline (SCNT)Genomic Sequence Confirmed and CRISPRs designed (1 day, if good EST data available)7-18-13 CRISPRs pairs ordered from IDT (1 day)7-19-13CRISPRs pairs annealed and ligated and transformedBacterial Colonies picked, propagated and sequence confirmedPositive colonies re-propagated in larger preps for transfections7/22/13-7/26/13 (1 week)Design Assays for smaller and large deletionsThis project had large assaysSmall assay 435 bp was developed (2 days)Cells ready for transfection 8-7-14First Transfection with CRISPRs (14 days)8/9/13-8/23/13Colonies picked and genotyped (2 days)8/23/14-8/24/14Positive Colonies were propagated and sequence confirmed 8-26-13 (this took along time)Nuclear Transfer 9/19/13Embryo Transfer 9/20/13First Pigs modified by CRISPRS 1/13/14

Total Time by SCNT: 5.5 - 6 months

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TimelinesEngineering Somatic Cells (1 month to 2 years)SCNT/ Zygote injection (1 week)Gestation (3 months, 3 weeks & 3 days)Puberty (6-9 months)

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Weaned wildtype and CD163 edited piglets prior to transport to Kansas State University.

Patent Pending

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Pigs Included in the Study3 piglets predicted to be null (CD163-/- )8 WT pigletsOne WT piglet humanely euthanized on day 1, due to poor body condition and not included in the studyK-State veterinarians and staff blinded to genotypes

Piglet IDPredicted TranslationMaternal AllelePaternal Allele#43CD163-/-7 bp addition in exon 72 bp deletion in exon 7 + 377 bp intron deletion in the preceding intron#55CD163-/-7 bp addition in exon 72 bp deletion in exon 7 + 377 bp intron deletion in the preceding intron#40CD163-/-7 bp addition in exon 711 bp deletion in exon 7

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Experimental DesignIdentity blinded to the crew at KSUHoused in the K-State BL-2 LARC facilityAllowed to acclimate for 3 days after arrival at facilityChallenge IM and IN with 105 TCID50 of NVSL 97-7985. (Standard lab isolate from 1997, relatively hot in terms of replication and pathogenesis)Pigs were maintained in the same pen; therefore, all pigs constantly exposed to virus Monitored daily for clinical signsBlood collected on days 0,4,7,11,14,21,28,35 and weights collected at least weeklyStudy terminated 35 days after infectionLung lavage for PAMsLungs and tissues removed for histopathology

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Clinical SignsFever was considered positive if it was 104FRespiratory scores ranged from:0: Normal, to 1: mild dyspnea and/or tachypnea when stressed (when handled), 2: mild dyspnea and/or tachypnea when at rest, 3: moderate dyspnea and/or tachypnea when stressed (when handled), 4: moderate dyspnea and/or tachypnea when at rest, 5: severe dyspnea and/or tachypnea when stressed (when handled), 6: severe dyspnea and/or tachypnea when at rest. Is there evidence of diarrhea (grade) or vomiting?

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Clinical Sign