Transgenic Animals - Technology and Applications

GOETZ LAIBLE, AGRESEARCHMWC - BIOLOGY TEACHERSPROFESSIONAL DEVELOPMENT DAYS6/7TH APRIL 2017

– Crown Research Institute in support of NZ’s pastoral industries

– Four major research centres • Ruakura• Grasslands• Lincoln• Invermay

• Total Staff 786 (full-time equivalent)

• Scientific/Technical Staff 405 FTE

• Science Groups• Animal Science (cattle, goats, sheep, deer)

• Food & Bio-based Products• Forage Science• Farm Systems & Environment• Knowledge & Analytics

AGRESEARCH – IN A NUTSHELL

Biomedical Biopharming Medical/functional foods Xenotransplantation Animal models of human diseases

Agricultural Improved quantity and quality of

animal production Improved animal health Sustainable agriculture

GM OF LIVESTOCK - A PLATFORM TECHNOLOGY FOR A VARIETY OF APPLICATIONS

Pronuclear Microinjection (1985)

TECHNOLOGICAL ADVANCES IN LIVESTOCK TRANSGENESIS

CLONING BY NUCLEAR TRANSFER

Wells et al., Biology of Reproduction, 1999Dolly the sheep, 1996

Pronuclear Microinjection

Somatic cell nuclear transfer (SCNT) with transfected cells (1997)

3. 2.

TECHNOLOGICAL ADVANCES IN LIVESTOCK TRANSGENESIS

Transfection of bovine cells

TransgenePromoter

GENERATION OF CLONED TRANSGENIC CATTLE

2.

Transfection of bovine cells

Oocyte enucleation

GENERATION OF CLONED TRANSGENIC CATTLE

3. 2.

Transfection of bovine cells

Oocyte enucleation

Fusion of donor cell and cytoplast

GENERATION OF CLONED TRANSGENIC CATTLE

3. 2.

Transfection of bovine cells

Oocyte enucleation

Fusion of donor cell and cytoplast

Activation of reconstructed

embryo

GENERATION OF CLONED TRANSGENIC CATTLE

3. 2.

Transfection of bovine cells

Oocyte enucleation

Fusion of donor cell and cytoplast

Embryo culture to blastocyst

Activation of reconstructed

embryo

GENERATION OF CLONED TRANSGENIC CATTLE

3. 2.

Transfection of bovine cells

Oocyte enucleation

Fusion of donor cell and cytoplast

Embryo culture to blastocyst

Activation of reconstructed

embryo

Embryo transfer

GENERATION OF CLONED TRANSGENIC CATTLE

Gene of InterestPromoter1) DNA construct

Secretion into milkActivated in the lactating mammary gland

4) Extraction of the pharmaceutical protein from milk and use as a drug for disease treatments

2) Stable integration of the DNA construct into the genome

3) Mammary gland produces large amounts of proteins that are readily accessible in milk

THE CONCEPT OF BIOPHARMING

Hereditary angioedema

BIOPHARMING – ALREADY A REALITY

rEVO Biologics2006 EMA/2009 FDA

Pharming NV2010 EMA/2014 FDA

ATryn® (antithrombin alpha)

Congenital antithrombindeficiency

RuconestTM (C1 esterase inhibitor)

KanumaTM (sebelipase alfa)

Alexion Pharmaceuticals2015 EMA/2015 FDA

Lysosomal acid lipase deficiency

Biosimilar antibody Cetuximab produced in the milk of goats

Erbitux approved for the treatment of metastatic colorectal cancer and head and neck cancer

LC

HC Puro

SCNT

GOATS PRODUCING AN ANTI-CANCER mAB

TG FOOD ANIMALS – A DIFFERENT STORY

Salmon with enhanced growth characteristics due to a growth hormone transgene

1989 – TG salmon generated

1995 – application for approval filed2009 – last supporting dataset submitted2010 – classified as safe to eat and safe for the environment and

recommended for approval

1993 – discussions with FDA initiated

– process stalled again and no final decision announced for

22 years in the regulatory maze

another 5 years

AquAdvantage salmon –approved for human consumption in November 2015

1ST TG FOOD ANIMALS APPROVED

Blocked by pending federal bill in March 2016 requesting new impact study on wild salmon stocks

ELIMINATING AN ALLERGY CAUSING PROTEIN PROTEIN

Daisy (with micro RNA)

BLG

Visualised milk proteins

Jabed et al., PNAS, 2012

Pronuclear Microinjection (1985)

Somatic cell nuclear transfer (SCNT) with transfected cells (1997)

ZFNs Zinc finger nucleases

TALENs Transcription activator-like effector nucleases

CRISPRs Clustered regularly interspaced short palindromic repeat (CRISPR) – Cas9 nucleases (2012)

GENETIC ENGINEERING ADVANCES IN LIVESTOCK

Genome Editing

*

* As of March 2017

Scientific publications mentioning CRISPR, 2007-2017

THE CRISPR REVOLUTION

ZFNs, TALENs and CRISPRs

Introduction of specific double strand breaks

TTTTTTTTTTTT

TTTTTTTTTTTT

small deletions small insertions

Non homologous end joining (NHEJ)

GENOME EDITING WITHOUT FOOTPRINT

CRISPR-mediated disruption (1506 bp deletion) of the CD163 gene

Whitworth et al., Nature Biotechnology, 2016

Whitworth et al., Biology of Reproduction, 2014

PRRSV-RESISTANT PIGS

WT

KO

(Procine Reproduction and Respiratory Syndrome Virus)

Rapid generation of elite genotypes by directly introducing beneficial natural mutations

Homologous recombination

T

TTTTT T

TTTTT

T

TTTTT T

TTTTT

T

TTTTT T

TTTTT

MOLECULAR BREEDING

HornlessHeat tolerant

ACCESSING THE BEST VARIANTS OF ALL GENES

Tan et al., PNAS, 2013

HORNLESS DAIRY CATTLE

Carlson et al., Nature Biotechnology 2015

Horned dairy cattle

Polled beef cattle

Polled dairy cattle

• A one amino acid change in ruminants prevents signal peptide cleavage of CD18 and generates binding site for bacterial leukotoxin

• Cell-mediated editing (correction) with ZFNs

Shanthalingam et al., PNAS 2016

SHIPPING FEVER-RESISTANT CATTLE

Introducing a ‘novel’ mutation

TUBERCULOSIS-RESISTANT CATTLE

Wu et al., PNAS 2015

Enhancing transgenesis with genome editing tools

ANIMALS FOR TRANSPLANT ORGANS

LARGE ANIMAL MODELS FOR HUMAN DISEASES

Mice have been the model of choice but differences in size and physiology to humans can be major shortcomings

Size, life span, physiology of large animals are more similar to humans

Enables study of chronic degenerative disease processes and testing of new therapeutic strategies and drugs

Cystic fibrosisCFTR KO and common human mutation ∆508Recapitulate devastating lung infections

LIVESTOCK MODELS OF HUMAN DISEASES

Huntington’s diseaseHTT transgene with 73 Q repeat

Rogers et al., Science 2008Pezzulo ey al., Nature 2012

Jacobsen et al., Hum Mol Genet 2010

Chris Slane, Farmers Weekly, 15 October 2012